MAKING AND USING IN VITRO-SYNTHESIZED ssRNA FOR INTRODUCING INTO MAMMALIAN CELLS TO INDUCE A BIOLOGICAL OR BIOCHEMICAL EFFECT

ABSTRACT

The present invention relates to compositions, kits and methods for making and using RNA compositions comprising in vitro-synthesized ssRNA inducing a biological or biochemical effect in a mammalian cell or organism into which the RNA composition is repeatedly or continuously introduced. In certain embodiments, the invention provides compositions and methods for changing the state of differentiation or phenotype of a human or other vertebrate cell. For example, the present invention provides mRNA and methods for reprogramming cells that exhibit a first differentiated state or phenotype to cells that exhibit a second differentiated state or phenotype, such as to reprogram human somatic cells to pluripotent stem cells.

The present application is a continuation of U.S. patent applicationSer. No. 16/225,489, filed Dec. 19, 2018, now allowed, which is acontinuation of U.S. patent application Ser. No. 14/368,399, filed Jun.24, 2014, now U.S. Pat. No. 10,201,620, issued Feb. 12, 2019, which is a§ 371 US National Entry Application of PCT/US2012/072301, filed Dec. 31,2012, which claims priority to the following applications: U.S.Provisional Application Ser. No. 61/582,050 filed Dec. 30, 2011; andU.S. Provisional Application Ser. No. 61/582,080 filed Dec. 30, 2011;U.S. Provisional Application Ser. No. 61/651,738 filed May 25, 2012;each of which are herein incorporated by reference as it fully set forththerein.

SEQUENCE LISTING

The text of the computer readable sequence listing filed herewith,titled “32359-306_SEQUENCE_LISTING_ST25”, created Mar. 15, 2022, havinga file size of 62,708 bytes, is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to RNA compositions, systems, kits, andmethods for making and using RNA compositions comprising invitro-synthesized ssRNA or mRNA to induce a biological or biochemicaleffect in human or other mammalian cells into which the RNA compositionis repeatedly or continuously introduced. In certain embodiments, thepresent invention pertains to RNA compositions and methods for makingand using the same for inducing biological or biochemical effects incells that are ex vivo in culture or cells that are in vivo in a tissue,organ or organism, wherein the biological effect may be induced in thecells, or in a tissue, organ or organism that contains the cells. Incertain embodiments, the RNA compositions are “substantially free,”“virtually free,” “essentially free,” “practically free,” “extremelyfree,” or “absolutely free” of dsRNA. In some embodiments, thebiological or biochemical effect comprises reprogramming cells thatexhibit a first differentiated state or phenotype to cells that exhibita second differentiated state or phenotype, such as to reprogram humansomatic cells to pluripotent stem cells, or to induce human fibroblastcells to neuron cells.

BACKGROUND

In 2006, it was reported (Takahashi and Yamanaka 2006) that theintroduction of genes encoding four protein factors (OCT4 (Octamer-4;POU class 5 homeobox 1), SOX2 (SRY (sex determining region Y)-box 2),KLF4 (Krueppel-like factor 4), and c-MYC) into differentiated mousesomatic cells induced those cells to become pluripotent stem cells,(referred to herein as “induced pluripotent stem cells,” “iPS cells,” or“iPSCs”). Following this original report, pluripotent stem cells werealso induced by transforming human somatic cells with genes encoding thesimilar human protein factors (OCT4, SOX2, KLF4, and c-MYC) (Takahashiet al. 2007), or by transforming human somatic cells with genes encodinghuman OCT4 and SOX2 factors plus genes encoding two other human factors,NANOG and LIN28 (Lin-28 homolog A) (Yu et al. 2007). All of thesemethods used retroviruses or lentiviruses to integrate genes encodingthe reprogramming factors into the genomes of the transformed cells andthe somatic cells were reprogrammed into iPS cells only over a longperiod of time (e.g., in excess of a week).

The generation iPS cells from differentiated somatic cells offers greatpromise as a possible means for treating diseases through celltransplantation. The possibility to generate iPS cells from somaticcells from individual patients also may enable development ofpatient-specific therapies with less risk due to immune rejection. Stillfurther, generation of iPS cells from disease-specific somatic cellsoffers promise as a means to study and develop drugs to treat specificdisease states (Ebert et al. 2009, Lee et al. 2009, Maehr et al. 2009).

Viral delivery of genes encoding protein reprogramming factors (or “iPSCfactors”) provides a highly efficient way to make iPS cells from somaticcells, but the integration of exogenous DNA into the genome, whetherrandom or non-random, creates unpredictable outcomes and can ultimatelylead to cancer (Nakagawa et al. 2008). New reports show that iPS cellscan be created (at lower efficiency) by using other methods that do notrequire genome integration. For example, repeated transfections ofexpression plasmids containing genes for OCT4, SOX2, KLF4 and c-MYC intomouse embryonic fibroblasts to generate iPS cells was demonstrated(Okita et al. 2008). Induced pluripotent stem cells were also generatedfrom human somatic cells by introduction of a plasmid that expressedgenes encoding human OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28 (Yu et al.2009). Other successful approaches for generating iPS cells includetreating somatic cells with: recombinant protein reprogramming factors(Zhou et al. 2009); non-integrating adenoviruses (Stadtfeld et al.2008); or piggyBac transposons (Woltjen et al. 2009) to deliverreprogramming factors. Presently, the generation of iPS cells usingthese non-viral delivery techniques to deliver reprogramming factors isextremely inefficient. Future methods for generating iPS cells forpotential clinical applications will need to increase the speed andefficiency of iPS cell formation while maintaining genome integrity.

SUMMARY OF THE INVENTION

The present invention relates to RNA compositions, systems, kits, andmethods for making and using RNA compositions comprising invitro-synthesized ssRNA or mRNA to induce a biological or biochemicaleffect in human or other mammalian cells into which the RNA compositionis repeatedly or continuously introduced. In certain embodiments, thepresent invention pertains to RNA compositions and methods for makingand using the same for inducing biological or biochemical effects incells that are ex vivo in culture or cells that are in vivo in a tissue,organ or organism, wherein the biological effect may be induced in thecells, or in a tissue, organ or organism that contains the cells.

In 2006, it was reported (Takahashi and Yamanaka 2006) that theintroduction of genes encoding four protein factors (OCT4 (Octamer-4;POU class 5 homeobox 1), SOX2 (SRY (sex determining region Y)-box 2),KLF4 (Krueppel-like factor 4), and c-MYC) into differentiated mousesomatic cells induced those cells to become pluripotent stem cells,(referred to herein as “induced pluripotent stem cells,” “iPS cells,” or“iPSCs”). Following this original report, pluripotent stem cells werealso induced by transforming human somatic cells with genes encoding thesimilar human protein factors (OCT4, SOX2, KLF4, and c-MYC) (Takahashiet al. 2007), or by transforming human somatic cells with genes encodinghuman OCT4 and SOX2 factors plus genes encoding two other human factors,NANOG and LIN28 (Lin-28 homolog A) (Yu et al. 2007). All of thesemethods used retroviruses or lentiviruses to integrate genes encodingthe reprogramming factors into the genomes of the transformed cells andthe somatic cells were reprogrammed into iPS cells only over a longperiod of time (e.g., in excess of a week).

The generation iPS cells from differentiated somatic cells offers greatpromise as a possible means for treating diseases through celltransplantation. The possibility to generate iPS cells from somaticcells from individual patients also may enable development ofpatient-specific therapies with less risk due to immune rejection. Stillfurther, generation of iPS cells from disease-specific somatic cellsoffers promise as a means to study and develop drugs to treat specificdisease states (Ebert et al. 2009, Lee et al. 2009, Maehr et al. 2009).

Viral delivery of genes encoding protein reprogramming factors (or “iPSCfactors”) provides a highly efficient way to make iPS cells from somaticcells, but the integration of exogenous DNA into the genome, whetherrandom or non-random, creates unpredictable outcomes and can ultimatelylead to cancer (Nakagawa et al. 2008). New reports show that iPS cellscan be created (at lower efficiency) by using other methods that do notrequire genome integration. For example, repeated transfections ofexpression plasmids containing genes for OCT4, SOX2, KLF4 and c-MYC intomouse embryonic fibroblasts to generate iPS cells was demonstrated(Okita et al. 2008). Induced pluripotent stem cells were also generatedfrom human somatic cells by introduction of a plasmid that expressedgenes encoding human OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28 (Yu et al.2009). Other successful approaches for generating iPS cells includetreating somatic cells with: recombinant protein reprogramming factors(Zhou et al. 2009); non-integrating adenoviruses (Stadtfeld et al.2008); or piggyBac transposons (Woltjen et al. 2009) to deliverreprogramming factors. Presently, the generation of iPS cells usingthese non-viral delivery techniques to deliver reprogramming factors isextremely inefficient. Future methods for generating iPS cells forpotential clinical applications will need to increase the speed andefficiency of iPS cell formation while maintaining genome integrity.

Immediately after disclosures by the laboratories of K. Yamanaka(Takahashi K et al., 2007) and JA Thomson (Yu J et al. 2007) reportinginduction of iPS cells from human somatic cells by viral or plasmidvectors which expressed genes encoding certain iPSC induction factors,one of the Applicants conceived that it might be possible to induceiPSCs by repeatedly transfecting human or animal somatic cells with invitro-synthesized mRNAs encoding such iPSC induction factors.

Introduction of in vitro-synthesized mRNA into eukaryotic cells andorganisms by means such as microinjection, electroporation andlipid-mediated transfection has been used to express encoded proteinssince the introduction of SP6, T7 and T3 in vitro transcription systemsabout 30 years ago (e.g., Krieg, P A and Melton, D A, 1984). Such work,usually involving one-time introductions into eukaryotic cells of anmRNA encoding a particular gene-encoded protein of interest, followed byassays and/or analyses of the proteins expressed, have yielded importantinformation about mRNA processing, the expression and activities ofgenes, and in vitro and in vivo translation of the encoded proteins.However, mRNA also was perceived to have certain disadvantages. Forexample, scientists perceive RNA to be more labile than DNA and believethat great care is needed to avoid degradation of RNA by a wide varietyof ubiquitous ribonucleases, as exemplified by RNases on human skin(Probst J et al., 2006).

Still further, many scientists have found that repeated transfection ofcells with in vitro-synthesized mRNA was cytotoxic to the cells andresulted cell death. For example, although Plews et al. (Plews J R etal., 2010) observed that pluripotency genes were activated upontransfection of human fibroblast cells with mRNAs encoding KLF4, c-MYC,OCT4 and SOX2 and LT proteins, they were unable to generate long-livediPSC lines, because, as they stated, “in all instances, very few cellssurvived and typically senesced within a week after treatment.” Whenthey also did brief treatments of the cells with certain small moleculessuch as valproic acid following mRNA transfection, they observedincreased activation of pluripotency genes compared to mRNA transfectionalone, but stated “during our attempt of multiple rounds ofmicroporation transfection, such treatment caused massive cell death.”Plews et al. also seemed to be skeptical of the results of Yakubov etal. (Yakubov et al., 2010), when they stated “Yakubov and colleaguesobtained similar AP positive colonies as us, however no differentiationanalyses were done, thus it is hard to evaluate the pluripotency of theiPS cells.”

Ugur Sahin et al. (Sahin U et al., 2011) also encountered great problemswith cytotoxicity and cell viability during attempts to reprogramsomatic cells to iPSCs with mRNA. After electroporating somatic cellswith ARCA-capped in vitro-transcribed mRNAs encoding the fourtranscription factors OCT4, SOX2, KLF4, and cMYC daily for multipledays, they observed that the mRNAs were translated and some markers foriPSCs were induced. However, they noted that “repetitive electroporationis associated with a loss of cell viability which became apparent onlyafter the second electroporation. The viability further decreased withevery following electroporation.” They attempted to “rescue” the cellsthat were being electroporated by continually adding more cells of thesame type as they were electroporating, but they did not state how theycould distinguish the previously electroporated cells from the new cellsamong the viable cells at the end of their electroporations. Apparently,they obtained no iPSC colonies that could be propagated ordifferentiated into other cells types, which are characteristics ofiPSCs, because they concluded their description of the experiment bystating that “The outgrowth of pluripotent colonies from these cells isstill under investigation.”

Similarly, in a recent paper on the repeated delivery of mRNAs encodingreprogramming factors KLF4, c-MYC, OCT4 and SOX2 into human fibroblasts,K Drews et al. (Drews K et al., 2012) reported that “upon repeatedtransfections, the mRNAs induced severe loss of cell viability asdemonstrated by MTT cytotoxicity assays. Microarray-derivedtranscriptome data revealed that the poor cell survival was mainly dueto the innate immune response triggered by the exogenous mRNAs. Wevalidated the influence of mRNA transfection on key immuneresponse-associated transcript levels, including IFNB1, RIG-I, PKR,IL12A, IRF7 AND CCL5, by quantitative PCR and directly compared thesewith levels induced by other methods previously published to mediatereprogramming in somatic cells.”

Such cytotoxicity and cell death as a result of repeated or continuousintroductions of in vitro-synthesized mRNA into cells may be due toinduction of RNA sensors and innate immune response mechanisms. Humanand animal cells possess wide array of RNA sensors and innate immuneresponse mechanisms that recognize and respond to exogenous RNAmolecules that may enter the cells, such as during viral or bacterialinfection. These cellular RNA sensors and innate immune responsemechanisms, if activated, can result in inhibition of protein synthesis,cytotoxicity, and programmed cell death via apoptotic signaling.

In support of this idea, Angel and Yanik (2010) showed that transfectionof cells with in vitro-synthesized mRNA activated innate immunity thatcaused significant cell death and that inhibition of innate immuneresponse genes using siRNA against IFN-beta, STAT2 and EIFAK2 (PKR)enabled frequent transfection of human fibroblasts with invitro-synthesized protein-encoding mRNA.

Kariko and Weissman (Kariko, et al., 2005; Kariko, et al., 2008; Kariko,et al., 2012) found that in vitro-synthesized modified mRNAs, in whichcanonical nucleosides were replaced by certain modified nucleosides(e.g. pseudouridine=ψ and e.g., 5-methylcytidine nucleosides=m⁵C), weremuch less immunogenic and were expressed into proteins at higher levelscompared to the corresponding in vitro-synthesized unmodified mRNAs.This work also supports the idea that the innate immune response needsto be reduced in order to express proteins encoded by repeatedlytransfected mRNA.

L. Warren et al. (Warren et al., 2010) reported reprogramming of humansomatic cells to iPSC colonies that could be continuously grown inculture and differentiated into cells comprising all 3 germ layers. Theydid this reprogramming by repeatedly transfecting somatic cells withARCA-capped phosphatase-treated (ψ and m⁵C)-modified mRNAs encoding KMOSor KMOSL transcription factors, where K=KLF4), M=MYC, O=OCT4, S=SOX2,L=LIN28, in medium containing B18R protein as an interferon inhibitor.Thus, Warren et al. used multiple methods to try to evade or counteractthe cellular RNA sensors and innate immune response mechanisms,including making the mRNA with two modified nucleotides which Kariko etal. had shown to result in a lower innate immune response, phosphatasingthe mRNA to remove the 5′triphosphate from the 20% of the mRNA moleculeswhich were not capped during the in vitro transcription reaction, andalso added B18R protein as an innate immune response inhibitor. Similarin vitro-synthesized mRNAs and methods, with some improvements, wereused in a subsequent publication (Warren et al., 2012).

Kariko et al. (Kariko et al., 2011A) disclosed expression of KMOSLNtranscription factors (N=NANOG) and reprogramming of human somatic cells(e.g., fibroblasts or keratinocytes) to iPSCs using mixtures of purifiedor treated in vitro-synthesized ψ-modified mRNAs (or mRNAs comprisingother modified nucleosides) encoding certain of these transcriptionfactors, without use of any added innate immune response inhibitor. Theuse of pseudouridine in place of uridine decreased the innate immuneresponse increased expression of the transcription factor proteinsencoded by the mRNA and, even then, purification or treatment of themRNA was necessary for successful reprogramming. This work furtherindicated that it was important and beneficial to evade or reduce theinnate immune response in order to decrease or eliminate cytotoxicityand cell death and induce reprogramming to iPSCs by repeatedlyintroducing protein-encoding mRNAs into somatic cells. The applicantsbelieve that it is critical for successful reprogramming or induction ofother biological or biochemical effects that in vitro-synthesized mRNAswhich are to be repeatedly or continuously introduced into human andanimal cells among other uses, must avoid inducing and activating thenumerous RNA sensors and innate immune response mechanisms that protectthem against pathogens comprising RNA.

However, in a recent paper, Lee et al. (Lee J, 2012), reviewed by L. A.J O'Neill (2012), argues just the opposite—that activation of innateimmunity by modified mRNA encoding KMOS proteins is required forefficient reprogramming of somatic cells to iPSCs. These authors believetheir data show that activation of toll-like receptor 3 (TLR3)-mediatedpathways (e.g., induction of type I IFN) is necessary for efficientinduction of pluripotency genes and induction of human iPSCs.

Resolution of this problem is important. Despite intense research, it isnot yet fully known in the art how or why cells recognize and tolerateendogenous mRNA molecules but do not tolerate repeated cellularintroduction of mRNA molecules synthesized by in vitro transcription,capping and polyadenylation. J. Eberwine and co-workers (Sul J-Y et al.,2012), who have focused on trying to use mRNA transcriptomes isolatedfrom cells to direct cell to cell phenotypic conversion, were perplexedby why scientists working on reprogramming using mRNA were encounteringproblems with cytotoxicity and cell death and using modified mRNA toreduce those effects, in view of the fact that they did not observesimilar effects using mRNA isolated from cells.

Thus, it is not understood what specific chemical and structuralfeatures of in vitro-synthesized mRNA are recognized by human ormammalian cellular RNA sensors to prevent such repeated cellularintroductions. Identifying these features and finding ways to be able torepeatedly or continuously introduce such in vitro-synthesized mRNAsinto human and animal cells would enable mRNA to be used to inducebiological or biochemical effects in cells, not only for reprogramming,but also for a wide variety of other important applications (e.g., forclinical research or for regenerative medicine or immunotherapy) in cellbiology, agriculture and medicine.

The reprogramming of human or animal somatic cells to iPSCs by repeatedor continuous transfection of in vitro-synthesized mRNAs encoding iPSCfactors provides an excellent model system for identifying whichfeatures of the in vitro-synthesized mRNAs are detected and whichcellular RNA sensors and innate immune response mechanisms inducecytotoxicity and cell death. Reprogramming is an excellent model becauseit requires daily transfections of multiple mRNAs over a period of about8 to about 18 days. Knowledge gained from reprogramming experiments willresult in easier, faster, more efficient and more effective cellularreprogramming, and also will likely lead to improved methods forinducing many other biological or biochemical effects ex vivo in cellsin culture or in vivo in cells in tissues, organs or organisms thatcontain them by repeated or continuous introduction of invitro-synthesized mRNA encoding one or more proteins.

Thus, the Applicants believe that methods and compositions developed forthis reprogramming model system may lead to: methods for making RNAcompositions comprising ssRNA for introduction into mammalian cells toinduce a biological or biochemical effect; new RNA compositions that aremore effective in inducing a biological or biochemical effect upon theirintroduction into mammalian cells; new methods for reprogramming cellsfrom a first state of differentiation or phenotype to a second state ofdifferentiation or phenotype (including dedifferentiation,transdifferentiation, and differentiation or re-differentiation); andnew methods for inducing other biological or biochemical effects inhuman or animal cells ex vivo in culture or in vivo in cells in tissues,organs or organisms by repeated or continuous introduction of invitro-synthesized mRNAs encoding one or more other proteins of interestinto the cells.

What is needed in the art is a better understanding of what specificchemical and structural features of in vitro-synthesized mRNAs arerecognized by cellular RNA sensors and innate immune response mechanismsto prevent repeated cellular introductions of the mRNAs. What is neededin the art are new methods, compositions and kits for making, purifyingand treating in vitro-synthesized mRNAs so that they can be repeatedlyor continuously introduced into human or animal (e.g., mammalian) cellsex vivo in culture or in human or animal (e.g., mammalian) cells in vivoin tissues, organs or organisms that contain the cells withoutactivating RNA sensors or inducing an innate immune response thatresults in significant cytotoxicity, cell death or inhibition of thedesired biochemical or biological effect for which the invitro-synthesized mRNAs are introduced into said cells. What is neededare new RNA compositions, new methods for making such RNA compositionscomprising in vitro-synthesized ssRNA or mRNA encoding one or moreproteins, methods for using such RNA compositions to repeatedly orcontinuously transfect human or animal (e.g., mammalian) cells in orderto cause a biological or biochemical effect (e.g., to reprogram a cellthat exhibits a first state of differentiation comprising a somatic cellto a cell that exhibits a second state of differentiation comprising aniPS cell) with higher efficiency and without inducing significantcytotoxicity or cell death.

Little or nothing is known about the results that could be obtained whensuch treated or purified RNA compositions are introduced into livingcells in culture or in human or animal subjects. What is needed in theart are better methods to generate RNA compositions comprising ssRNA ormRNA for repeated or continuous introduction into cells ex vivo inculture or in vivo in human or animal subjects (e.g., for biological andclinical research, agriculture or clinical applications).

Repeated or continuous introduction of mRNA into cells to induce abiological or biochemical effect (e.g., for reprogramming) may providebenefits over introduction of DNA or protein molecules. For example,introduction of mRNA into a cell is less likely than DNA to result ingenome insertions or genetic modifications, with related permanenteffects for the cells. Also, it may be easier to introduce mRNA into acell, wherein it is properly post-translationally modified for optimalexpression, than to make and deliver proteins with a particularglycosylation or other post-translational modification appropriate forthe particular cell. Thus, what is needed are effective methods formaking, for repeatedly or continuously introducing, and for expressingmRNA in living cells to induce biological or biochemical effects (e.g.,in the biologic, agricultural and clinical fields of use, e.g., for usein regenerative medicine, cell reprogramming, cell-based therapies,enzyme replacement therapies, cell, tissue and organ transplantation orrepair, tissue or organ engineering, and immunotherapies).

In certain embodiments, the present invention pertains to embodiments ofcompositions, reaction mixtures, kits and methods that comprise or useone or more in vitro-synthesized single-stranded RNAs (ssRNAs) ormessenger RNAs (mRNAs) (sometimes also referred to as ssRNA or mRNAmolecules). With respect to the present invention, an “invitro-synthesized ssRNA or mRNA” herein means and refers to ssRNA ormRNA that is synthesized or prepared using a method comprising in vitrotranscription of one or more DNA templates by an RNA polymerase. Stillfurther, unless specifically stated otherwise, the terms “ssRNA” or“mRNA” when used herein with reference to an embodiment of the presentinvention shall mean an “in vitro-synthesized ssRNA or mRNA” as definedabove. In preferred embodiments, the in vitro-synthesized ssRNA or mRNAencodes (or exhibits a coding sequence of) at least one protein orpolypeptide. In some preferred embodiments, the ssRNA or mRNA encodes atleast one protein that is capable of effecting a biological orbiochemical effect when repeatedly or continuously introduced into ahuman or animal cell (e.g., a mammalian cell). In some preferredembodiments, the invention comprises a composition comprising ssRNA ormRNA, and, unless specifically stated otherwise, the term “RNAcomposition” shall mean an RNA composition comprising or consisting ofin vitro-synthesized ssRNA or mRNA. In some preferred embodiments, theinvention comprises an RNA composition comprising or consisting of invitro-synthesized ssRNA or mRNA that encodes one protein or polypeptide.

In some preferred embodiments, the invention comprises an RNAcomposition comprising or consisting of a mixture of multiple differentin vitro-synthesized ssRNAs or mRNAs, each of which encodes a differentprotein. Other embodiments of the invention comprise an RNA compositioncomprising or consisting of in vitro-synthesized ssRNA that does notencode a protein or polypeptide, but instead exhibits the sequence of atleast one long non-coding RNA (ncRNA). Still other embodiments comprisevarious reaction mixtures, kits and methods that comprise or use an RNAcomposition.

One embodiment of the present invention is a method for treating invitro-synthesized ssRNA or mRNA to generate an RNA composition that is“substantially free of dsRNA,” “virtually free of dsRNA,” “essentiallyfree of dsRNA,” “practically free of dsRNA,” “extremely free of dsRNA,”or “absolutely free of dsRNA,” meaning, respectively, that less thanabout: 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or 0.0002% of the mass of theRNA in the treated ssRNA composition is dsRNA of a size greater thanabout 40 basepairs, (or greater than about 30 basepairs) the methodcomprising: contacting the in vitro-synthesized ssRNA or mRNA with RNaseIII protein in a buffered aqueous solution comprising magnesium cationsat a concentration of about 1-4 mM; and a salt providing an ionicstrength at least equivalent to about 50 mM potassium acetate orpotassium glutamate, and incubating under conditions wherein the RNAcomposition is generated.

Thus, one embodiment of the present invention is a method for treatingin vitro-synthesized ssRNA or mRNA to generate a treated RNA compositionwherein less than about: 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or 0.0002%,respectively, of the mass of the RNA in the treated RNA composition isdsRNA of a size greater than about 40 basepairs (or greater than about30 basepairs), the method comprising: contacting the invitro-synthesized ssRNA or mRNA with RNase III protein in a bufferedaqueous solution comprising magnesium cations at a concentration ofabout 1-4 mM; and a salt providing an ionic strength at least equivalentto about 50 mM potassium acetate or potassium glutamate, and incubatingunder conditions wherein the treated RNA composition is generated.However, unless otherwise obvious from the description or otherwisespecifically stated, whenever we say that an “RNA composition” is usedin a method described herein wherein the RNA composition is repeatedlyor continuously contacted with or introduced into a human or animal cell(e.g., a mammalian cell) to induce a biological or biochemical effect(e.g., to reprogram a cell that exhibits a first differentiated state orphenotype to a second differentiated state or phenotype), we mean (andit will be understood) that said RNA composition is either a treated RNAcomposition that was generated using the presently described method, oris a purified RNA composition wherein less than: 0.01%, 0.001% or0.0002% (or a specifically stated percentage) of the mass of the RNA inthe purified RNA composition is dsRNA of a size greater than about 40basepairs (or greater than about 30 basepairs), even when said RNAcomposition is not referred to as a “treated RNA composition” or a“purified RNA composition.”

One embodiment of the invention is an RNA treatment reaction mixturecomprising: a) an in vitro-synthesized ssRNA or mRNA (e.g., that encodesone or more proteins or one or more long non-coding RNAs (ncRNAs); b) adouble-stranded RNA (dsRNA)-specific endoribonuclease III (endoRNase IIIor RNase III) protein; c) magnesium cations at a concentration of about1-4 mM; and d) a salt providing an ionic strength at least equivalent to50 mM potassium acetate or potassium glutamate; wherein said RNAtreatment reaction mixture is practically free, extremely free orabsolutely free of dsRNA, meaning that less than 0.01%, less than 0.001%or less than 0.0002%, respectively, of the RNA in the RNA treatmentreaction mixture is dsRNA of a size greater than about 40 basepairs (orgreater than about 30 base pairs).

Prior to the present invention, said RNA treatment reaction mixture andsaid method for making a treated RNA composition wherein less than0.01%, less than 0.001% or less than 0.0002% of the RNA in the RNAtreatment reaction mixture or RNA composition was dsRNA of a sizegreater than about 40 basepairs were not known in the art, as evidencedby the EXAMPLES disclosed herein. For example, treatment of an RNAcomposition comprising in vitro-transcribed unmodified GAUC ssRNA (e.g.,mRNAs encoding iPSC induction factors) using RNase III as described inthe art (e.g., Robertson, 1968) did not generate a treated RNAcomposition that resulted in reprogramming human fibroblasts to iPSCswhen the treated RNA composition was repeatedly introduced into thefibroblast cells (e.g., see reprogramming results using RNase IIItreatments with 10 mM magnesium acetate in the Table in EXAMPLE 10),whereas the RNase III treatment method of the present invention didresult in successful reprogramming (e.g., see reprogramming resultsusing RNase III treatments with about 1-4 mM magnesium acetate in theTable in EXAMPLE 10). This surprising and unexpected result was furtherexplained by the results of other experiments (e.g., see EXAMPLE 22).For example, the Table in EXAMPLE 22 shows that the addition of dsRNA ata level of only about 0.001% or more of the total RNA in an RNAcomposition comprising a mixture of highly purified unmodified ssRNAs(e.g., mRNAs encoding iPSC induction factors) is sufficient toeffectively inhibit reprogramming of human fibroblasts in culture toiPSCs.

Thus, some embodiments of the method for treating in vitro-synthesizedssRNA or mRNA, generate an RNA composition that is substantially free,virtually free, essentially free, practically free, extremely free orabsolutely free of dsRNA,

Preferred embodiments of the present invention comprise RNA compositionscomprising ssRNA or mRNA that are at least practically free ofdouble-stranded RNA (e.g., practically, extremely or absolutelydsRNA-free compositions), methods and kits for making at leastpractically dsRNA-free compositions, and kits and methods comprisingand/or for using at least practically dsRNA-free compositions.

One particular embodiment of the invention is an RNA composition that isat least practically free of dsRNA, wherein said RNA compositioncomprises in vitro-synthesized ssRNA (e.g., ncRNA or mRNA (e.g.,encoding one or more proteins), and wherein said RNA composition is:“practically free of dsRNA,” “extremely free of dsRNA,” or “absolutelyfree of dsRNA,” meaning, respectively, that less than: 0.01%, 0.001%, or0.0002% of the RNA in the RNA composition comprises dsRNA of a sizegreater than about 40 basepairs. For example, one particular embodimentof the invention is an RNA composition comprising one or more invitro-synthesized ssRNAs or mRNAs encoding one or more proteintranscription factors, wherein the RNA composition is practically free,extremely free or absolutely free of dsRNA. Another RNA composition ofthe invention is a reaction mixture comprising an RNA treatment reactionmixture comprising: a) an in vitro-synthesized ssRNA or mRNA thatencodes one or more proteins transcription factors; b) a double-strandedRNA (dsRNA)-specific endoribonuclease III (endoRNase III or RNase III)protein; c) magnesium cations at a concentration of about 1-4 mM; and d)a salt providing an ionic strength at least equivalent to 50 mMpotassium acetate or potassium glutamate; wherein said RNA treatmentreaction mixture is practically free, extremely free or absolutely freeof dsRNA, meaning that less than 0.01%, less than 0.001% or less than0.0002%, respectively, of the RNA in the RNA treatment reaction mixtureis dsRNA of a size greater than about 40 basepairs.

In some embodiments, the amounts and relative amounts of dsRNA tonon-contaminant ssRNA or mRNA is determined using a dsRNA-specificantibody as described herein. In some embodiments, the amounts andrelative amounts of non-contaminant mRNA molecules and RNA contaminantmolecules (or a particular RNA contaminant) may be determined by HPLC orother methods used in the art to separate and quantify RNA molecules.

Thus, the present invention provides methods for synthesizing an invitro transcribed (IVT) RNA composition, and then contacting the IVT RNAcomposition with a dsRNA-specific RNase, such as RNase III, underconditions wherein contaminant dsRNA can be reproducibly digested andssRNA molecules that do not induce or activate a dsRNA innate immuneresponse pathway or RNA sensor can reliably be generated.

When the Applicants attempted to use RNase III as described in the art(e.g., by Robertson et al., 1968, and by Mellits et al., 1990) as apotential solution to treat ssRNA comprising mRNA molecules fortranslation in living cells, the Applicants were surprised to find thatthe RNase III-treated ssRNAs were toxic. Thus, human cells that weretransfected with various doses of the RNase III-treated ssRNAs, eitherdaily or every-other-day for up to 3 weeks, appeared increasingly lesshealthy during the course of said introducing and finally died. Further,the Applicants found that RNase III-treated ssRNAs obtained using theprotocol originally described by Robertson et al. remained contaminatedwith significant amounts of dsRNA based on dot-blot immunoassays usingtwo different dsRNA-specific antibodies (J2 and K1 antibodies; Englishand Scientific Consulting, Szirák, Hungary).

Accordingly, the Applicants found that RNase III-treated ssRNA preparedas described in the art could not be introduced into human cells for invivo translation. Still further, extending the reaction incubation timeof the RNase III reaction also did not noticeably reduce the toxicity ofthe ssRNA to the cells or reduce the amount of contaminant dsRNA tobelow the detection levels of the dsRNA-specific antibodies. Increasingthe reaction time also appeared to result in greater degradation of thessRNA, based on staining of electrophoresis gels, and less expression ofthe ssRNA.

Mellits et al. (1990) provided guidance related to the RNase IIIprotocol that it may be necessary to “optimize the digestion conditionswith respect to enzyme/substrate ratio, salt concentration, andtemperature for a particular RNA.”

Accordingly, the present Applicants modified the RNase III protocol byvarying the amount of RNase III relative to a constant amount of RNAtreated. However, increasing or decreasing the amount of enzyme relativeto the amount of RNA did not affect the amount of dsRNA that remainedafter the protocol.

Next, the present Applicants carefully evaluated whether changing theconcentration or type of monovalent salt (including other salts than theNH₄Cl salt taught with respect to the standard Robertson RNase III assayprotocol would positively affect the results. Provided that themonovalent salt concentration was sufficient to maintain the duplexstate of the dsRNA (e.g., at least 50 mM or greater, the differentconcentrations did not result in increased digestion of the contaminantdsRNA molecules. At high monovalent salt concentrations, there appearedto be a slight inhibition of RNase III activity. Longer RNase IIIreaction times or higher reaction temperatures appeared to increasedegradation of the ssRNA of interest without increasing digestion of thecontaminating dsRNA.

The present Applicants next designed an RNA substrate comprising bothsingle-stranded portions and a double-stranded portion in order to moreaccurately and precisely evaluate both the dsRNA-specific activity andthe specificity of digestion for dsRNA rather than ssRNA since thevarious RNase III reaction conditions could be assayed using this singlesubstrate (FIG. 1). As shown in FIG. 1, correct digestion of this RNAsubstrate would be expected to result in complete digestion of thecentral 1671-basepair dsRNA portion, while leaving ssRNA tails of 136bases and 255 bases intact. This substrate turned out to be a valuabletool in the present studies.

Using this substrate, very surprisingly and unexpectedly, the presentApplicants discovered a dramatic improvement in both the RNase IIIactivity and specificity when the concentration of divalent magnesiumcations was decreased by about 10-fold compared to the concentrationtaught in the art (e.g., Robertson et al., 1968). Thus, at aconcentration of 1 mM divalent magnesium cations, the single-strandedtails of the substrate remained intact and the dsRNA central portion wascompletely digested. The substrate was then used to precisely titratethe optimal range of divalent magnesium concentration. Surprisingly,whereas the literature (e.g., Robertson et al., 1968) had reported that“[m]agnesium stimulates activity over a broad range between 0.005 M and0.1 M), the present Applicants found that it was necessary to use aconcentration of divalent magnesium of about 4 mM or less, andpreferably about 1-3 mM, or 1-2 mM) in order to sufficiently digest thedsRNA so that the RNA composition comprising ssRNA molecules did notinduce or activate a dsRNA-specific innate immune response or RNA sensorpathways that resulted in a substantial decrease in protein synthesis,increase in cell toxicity, or cell death. Still further, at this lowermagnesium cation concentration range, the yield of intact ssRNAmolecules increased. Both of these effects—decreased levels of dsRNA andincreased levels of intact ssRNA—resulted in higher levels oftranslation of mRNAs encoding a variety of different proteins comprisingreprogramming factors and much less toxicity to the cells, as reflectedby much lower levels of cellular expression of a various innate immuneresponse-related genes (based on quantitative RT-PCR analysis).

Specifically, the RNase III protocol taught in the art since about 1968has taught to use magnesium acetate at 10 mM. However, the presentApplicants found that a 10 mM concentration of magnesium acetateresulted in toxicity to the cells due to induction and/or activation ofa strong innate immune response. However, surprisingly and unexpectedly,the present Applicants found that treating the ssRNA with RNase III in areaction mixture comprising only about 1-4 mM, and more preferably about1-3 mM magnesium acetate, resulted in ssRNA that was intact (withoutnoticeable smearing of the ssRNA band on electrophoresis) and with muchless dsRNA (which we much later determined to be at least practicallyfree, extremely free or absolutely free of the dsRNA), which ssRNA alsoresulted in much less toxicity and cell death when repeatedly introducedinto human or animal cells.

Evidence for the more complete digestion of dsRNA, while bettermaintaining the integrity of the ssRNA during the RNase III digestion isshown in EXAMPLE 1 and FIG. 2. As shown in FIG. 2, at magnesium acetateconcentrations between 1 and 4 mM, the 1671-basepair dsRNA region of theRNA substrate was completely digested and the two ssRNA fragments of 255and 136 nucleotides remained intact. At a concentration of 5 mMmagnesium acetate, the ssRNAs were noticeably more degraded, as seen bythe smear under the ssRNA bands, and this degradation increased as themagnesium acetate concentration increased to from 6 to 10 mM magnesiumacetate, with very significant smearing at 10 mM.

Dot blot assays of digestion of varying amounts of dsRNA by RNase III inthe presence of different concentrations of divalent magnesium acetateusing a dsRNA-specific antibody, as shown in EXAMPLE 2 and EXAMPLE 3(FIG. 3 and FIG. 4, respectively) confirmed that the dsRNA was mosteffectively digested by the RNase III treatment in a reaction mixturecomprising a final concentration of between about 1 mM and about 4 mMmagnesium acetate, and more preferably, between about 2 mM and about 4mM magnesium acetate. When the RNase III treatment of invitro-synthesized ssRNA was performed at this concentration range, thepresent Applicants found in other experiments that the toxicity of thetreated ssRNA upon repeated daily transfection into cells wassignificantly reduced compared to ssRNA treated at magnesium cationconcentrations higher than 4 mM (e.g., at 10 mM as taught by Mellits etal., 1990), and this reduction in toxicity of the ssRNA during repeatedtransfections was critical to be able to successfully reprogram humansomatic cells to induced pluripotent stem (iPS) cells.

Accordingly, the method developed by the present Applicants was found tobe essential, effective, and reproducible for achieving successfulreprogramming of human somatic cells using ssRNAs encoding reprogrammingfactors. The method is capable of treating both small and largequantities of RNA by removing dsRNA contaminants generated during invitro transcription while maintaining the integrity of the ssRNA.

The method has been shown to be unexpectedly successful in reducinginduction and/or activation of innate immune response signaling pathwaysand RNA sensors (e.g., TLR3-mediated interferon induction) in humancells in response introducing in vitro-synthesized ssRNA into the cells,even after multiple (e.g., daily) transfections for up to about 21 days.For example, if no purification or RNase III treatment is performed toremove dsRNA, it is not possible to successfully reprogram BJfibroblasts to iPS cells. This is because even minute quantities ofcontaminating dsRNA, when transfected every day for multiple days (e.g.,daily for >2 days, >3 days, >5 days, >8 days, >10 days, >12 days, >14days, >16 days, >18 days, or >20 days) results in high toxicity to thecells. For example, the present Applicants have observed that most orall of the fibroblast cells die if transfected for more than about 6 toabout 10 days with in vitro-transcribed mRNAs encoding iPSC inductionfactors which have not been purified or treated to remove the dsRNA(with survival time depending upon the dose of ssRNAs transfected, theparticular cells, the transfection reagent or method used, and otherfactors). However, by using the presently-described RNase III treatmentmethod comprising use of about 1 mM to about 4 mM of divalent magnesiumcations to digest dsRNA contaminant molecules in in vitro-synthesizedssRNA (e.g., mRNA), thereby reducing the TLR3-mediated innate immuneresponse, it was possible to efficiently reprogram human BJ fibroblaststo induced pluripotent stem cells (iPSCs) by transfecting the cells withRNase III-treated unmodified ssRNAs comprising cap1 5′-capped mRNAshaving approximately 150-base poly(A) tails, which mRNAs encoded iPSCinduction factors, daily for up to 18 days (e.g., see EXAMPLE 10); incontrast, no reprogramming of BJ fibroblasts to iPSCs was observed inEXAMPLE 10 when the same unmodified ssRNAs were treated with RNase IIIin the presence of 10 mM divalent magnesium cations. Still further,unmodified ssRNAs treated with RNase III in the presence of 1-4 mMdivalent magnesium cations resulted in much less toxicity and death ofthe BJ fibroblasts compared to the same unmodified ssRNAs treated withRNase III in the presence of 10 mM divalent magnesium cations. Forexample, in this particular experiment, this is a main factor for whygreater than 100 iPSCs were induced in BJ fibroblasts transfected everyday for 13 days with the 1.2 micrograms of a 3:1:1:1:1:1 molar mix ofthe unmodified ssRNAs encoding OCT4, SOX2, KLF4, LIN28, NANOG, andcMYC(T58A), respectively, that was treated with RNase III in thepresence of 1 mM divalent magnesium cations and 200 mM potassium acetateas the monovalent salt, whereas no reprogramming of BJ fibroblasts toiPSCs was observed if the same unmodified ssRNAs were treated with RNaseIII in the presence of 10 mM divalent magnesium cations. Those withknowledge in the art will especially recognize the power of the presentRNase III treatment method to prepare in vitro-transcribed ssRNA that iscapable of inducing a biological or biochemical effect upon repeated orcontinuous introduction into cells in view of the fact that, it isbelieved that, prior to the work described herein, no one had reportedor described in the art the use of unmodified GAUC mRNAs encoding iPSCfactors to reprogram somatic cells to iPS cells which could be growninto iPS cell lines and differentiated into other types of cellsrepresenting all three germ layers (as described herein). Thus, theRNase III treatment method described herein provides, for the firsttime, a simple and straightforward method to remove even minutequantities of contaminating dsRNA from in vitro-synthesized mRNA,thereby successfully solving the problem of cell toxicity and cell deaththat results from using unpurified or untreated in vitro-synthesizedmRNA.

As disclosed in Kariko et al. (Kariko et al., 2011), Drs. Weissman andKariko, showed that HPLC could be used to purify in vitro-synthesizedmRNA comprising modified nucleotides, such as pseudouridine or bothpseudouridine and 5-methylcytidine, and, working with the presentApplicants, showed that HPLC-purified modified mRNAs encoding iPSCinduction factors could be used to reprogram somatic cells to iPS cells.The present Applicants show herein that the RNase III treatment methoddisclosed herein is approximately equivalent to HPLC purification forremoving dsRNA from in vitro-synthesized mRNA based on a quantitativecomparison of the number of iPS cells induced from fibroblasts usingiPSC induction factor-encoding modified mRNAs purified by HPLC ortreated with the RNase III treatment described herein (e.g., see tablesin the Results for EXAMPLE 15 and EXAMPLE 27). While the presentinvention is not limited to any particular mechanism or theory, and anunderstanding of the mechanism or theory is not necessary tosuccessfully practice the present invention, since mRNAs were purifiedas single peaks by HPLC, our finding that HPLC-purified and RNaseIII-treated mRNAs appear to be quantitatively equivalent in inducing iPScells from somatic cells strongly suggests that dsRNA generated duringthe in vitro transcription reaction is the sole contaminant in the CAP1poly(A)-tailed pseudouridine-modified mRNAs that induced the innateimmune responses that we observed if the mRNAs were not purified by HPLCor treated using the presently-described RNase III treatment. Stillfurther, in view of the equivalence of the RNase III treatment to HPLCin terms of removing the dsRNA contaminant, those with skill in the artwill recognize the advantages and benefits of the RNase III treatmentmethod over HPLC purification. For example, the RNase III treatmentmethod described herein does not require scientists to learn how tooperate and purchase expensive equipment, columns, and reagents, anddoes not require washing of columns, or generate organic solvent waste,as does HPLC. The RNase III treatment is also much faster and easierthan HPLC, requiring minimum hands-on time and only about 30 minutes forthe treatment itself, plus a small amount of additional time for organicextraction, ammonium acetate precipitation, ethanol washes of theprecipitate, followed by storage as a dry pellet or, if desired,suspension in an aqueous solution. When performed as described for thestandard RNase III treatment, the Applicants have found the method to beextremely reliable and reproducible with at least a couple of dozendifferent mRNAs. For example, the Applicants have used the RNase IIItreatment method routinely for preparation of mRNAs encoding differenttranscription factors that were repeatedly or continuously transfectedinto human or animal cells for use in reprogramming the cells from onestate of differentiation to another, without encountering unexpectedproblems. The Applicants were surprised that, as described in EXAMPLE23, the RNase III treatment was necessary for reprogramming of mousemesenchymal stem cells to myoblast cells using modified mRNA encodingMYOD. Thus, even though only two daily transfections were needed for thereprogramming using mRNA prepared using the RNase III treatment, nomyoblasts were induced by mRNA encoding MYOD which had not been preparedusing the presently-described RNase III treatment. This indicates thatRNA sensors or innate immune responses can inhibit a desired biologicalor biochemical effect even when only a short amount of time and a smallnumber of transfections are needed.

The Applicants have also used the RNase III treatment method to prepareother mRNAs for repeated or continuous transfection into human or animalcells in order to induce biological or biochemical effects other thanreprogramming of cells from one state of differentiation to another, andhave found that the resulting RNase III-treated mRNAs were less toxicand were translated into protein at higher levels than the same mRNAsthat were not RNase III-treated.

In general, due to the simplicity of the protocol, the RNase IIItreatment method can also be used to treat many in vitro-synthesizedRNAs simultaneously in parallel and, since it involves simple steps,such as pipetting, the method is also capable of being automated by useof a robot, or scaled up for treatment of any desired amount of RNA.

If capping and polyadenylation of in vitro-transcribed ssRNAs is donepost-transcriptionally using a capping enzyme comprising RNAguanyltransferase and a poly(A) polymerase, preferably the RNase IIItreatment is performed after the in vitro transcription and beforecapping and polyadenylation. However, we have also achieved good results(e.g., for reprogramming somatic cells to iPSCs) when the RNase IIItreatment was applied to ssRNAs after capping or polyadenylation. Asshown herein, the RNase III treatment was also successful for removingdsRNA from in vitro-transcribed ssRNA that was cappedco-transcriptionally using a dinucleotide cap analog (e.g., an ARCA)and/or polyadenylated during in vitro transcription of a DNA templatethat also encoded the poly(A) tail.

As discussed above and elsewhere herein, reprogramming of fibroblasts toiPS cells using unmodified or pseudouridine modified invitro-transcribed ssRNA was not observed unless the ssRNA was purified(e.g., by a method such as chromatography (e.g., HPLC), electrophoresis,or treated using the presently described RNase III treatment). Withoutbeing bound by theory, we believe that this is because even minutequantities of contaminating dsRNA, when transfected every day for 18days, would result in high toxicity to the cells. For example, evenminute quantities of contaminating dsRNA induce high levels of type Iinterferons, which in turn inhibit translation in the cells in aPKR-dependent mechanism. Further, the type I interferons inducethousands of genes to defend the cells against invasion by the dsRNA,which is the same mechanism that the cell uses to protect itself againstpathogenic dsRNA viruses. Still further, it has been reported that typeI and type II interferons can sensitize cells to dsRNA-inducedcytotoxicity, which might tip the balance from necrosis to apoptosis(Stewart II, W E et al., 1972; Kalai, M et al., 2002). Thus, the factthat the ssRNAs are introduced into the cells every day for multipledays (e.g., up to 18 or more days to induce iPS cells) may be animportant factor in cytotoxicity and apoptosis. The innate immuneresponse is induced, leading to interferon production, which in turncauses protein translation to be decreased or shut down for a longertime, and eventually, the apoptotic signaling pathways are activated,leading to cell death.

Thus, we believe the presently described methods are important becausethey reduce the levels of contaminating dsRNA so that the purified ortreated ssRNAs can be introduced into the cells without inducingcytotoxicity and cell death, including wherein the purified or treatedssRNAs are repeatedly introduced into the living human or animal cells(e.g., daily for multiple days or multiple weeks for cells in cultureor, potentially, daily or weekly for multiple weeks, months or evenyears when introduced into cells in a human or animal organism).

In some embodiments, the RNase III treatment methods are useful forpreparing any ssRNA for translation or expression in human or animalcells, and can be performed on multiple samples simultaneously in lessthan one hour, with only minutes of hands-on time. Due to the simplicityof the methods, they are also amenable to automation and scale-up (e.g.,for high-throughput applications).

Surprisingly and unexpectedly, when this method was used to generatedtreated ssRNAs from in vitro-synthesized ssRNAs comprising or consistingof either only unmodified ribonucleosides (G,A,C,U), or Ψ- and/orm⁵C-modified ribonucleosides that encoded iPSC induction factors (e.g.,OCT4, SOX2, KLF4, LIN28, NANOG and either c-MYC, c-MYC(T58A), or L-MYC),the treated ssRNAs were highly efficient in reprogramming human somaticcells (e.g., fibroblasts or keratinocytes) to pluripotent stem cells(iPSCs) when introduced into the cells once daily for ˜10 to ˜21 days,without using any agent that reduces the expression of proteins in aninnate immune response pathway (e.g., without B18R protein). Aftermaking stable iPSC lines (meaning cell lines which maintained iPSC cellmarkers and the ability to differentiate into cells of all 3 germ layersover an extended period of time) from iPSC colonies, they were confirmedto be iPSCs based on immunostaining for iPSC markers and weredifferentiated into cells representing all three germ layers using anembryoid body differentiation assay. Induction of iPSCs using ssRNAswithout an inhibitor or agent (e.g., B18R protein) that reduces theexpression of an innate immune response pathway or using ssRNAconsisting of only unmodified canonical ribonucleosides has not beenreported by others, it is believed, and clearly shows the power of themethod for making treated protein-encoding ssRNA for translation inhuman or animal cells.

In certain embodiments, the ssRNAs treated using RNase III comprise oneor more different ssRNA molecules that are treated with RNase III enzymein a reaction buffer comprising divalent magnesium cations at a finalconcentration of about 1 mM to about 4 mM. In certain preferredembodiments, one or more different ssRNAs are treated using an RNase IIItreatment method comprise with RNase III enzyme in a reaction buffercomprising divalent magnesium cations at a final concentration of about1 to about 3 mM, more preferably about 1 mM, about 2 mM or about 3 mM.In some embodiments, the method generates ssRNA that is substantiallyfree of dsRNA, meaning that, after the RNase III treatment and cleanup,greater than about 99.5% of the RNA is ssRNA and less than about 0.5% ofthe RNA is dsRNA greater than about 40 bp (or greater than about 30 bp).In some embodiments, the method generates ssRNA that is virtually freeof dsRNA, meaning that, after the RNase III treatment and cleanup,greater than about 99.9% of the RNA is ssRNA and less than about 0.1% ofthe RNA is dsRNA greater than about 40 bp (or greater than about 30 bp).In some embodiments, the method generates ssRNA that is essentially freeof dsRNA, meaning that, after the RNase III treatment and cleanup,greater than about 99.95% of the RNA is ssRNA and less than about 0.05%of the RNA is dsRNA greater than about 40 bp (or greater than 30 bp). Insome embodiments, the method generates ssRNA that is practically free ofdsRNA, meaning that, after the RNase III treatment and cleanup, greaterthan about 99.99% of the RNA is ssRNA and less than about 0.01% of theRNA is dsRNA greater than about 40 bp (or greater than 30 bp). In someembodiments, the method generates ssRNA that is extremely free of dsRNA,meaning that, after the RNase III treatment and cleanup, greater thanabout 99.999% of the RNA is ssRNA and less than about 0.001% of the RNAis dsRNA greater than about 40 bp (or greater than about 30 bp). In someembodiments, the method generates ssRNA that is absolutely free ofdsRNA, meaning that, after the RNase III treatment and cleanup, greaterthan about 99.9998% of the RNA is ssRNA and less than about 0.0002% ofthe RNA is dsRNA greater than about 40 bp (or greater than about 30 bp).

In one embodiment, the dsRNA-specific RNase is RNase III and the methodcomprises treating in vitro-synthesized ssRNAs with the RNase III in areaction mixture comprising divalent magnesium cations at aconcentration of about 1 mM to about 4 mM, and then removing the RNaseIII digestion products and reaction mixture components to generate thetreated ssRNAs that are substantially, virtually, essentially,practically, extremely or absolutely free of dsRNA.

In certain preferred embodiments, the RNase III-treated ssRNAs generatedusing the methods do not result in an innate immune response thatresults in substantial inhibition of cellular protein synthesis ordsRNA-induced apoptosis after introducing the treated ssRNAs into thecells at least two times or at least three times. In one preferredembodiment, the one or more in vitro-synthesized ssRNAs encode inducedpluripotent stem cell (iPSC) induction factors, the cells that exhibit afirst differentiated state are human or animal somatic cells, and thetreated ssRNAs or purified ssRNAs are introduced into said cells on eachof about 15 to about 21 days (e.g., 15, 16, 17, 18, 19, 20, or 21 days)to generate cells that exhibit a second differentiated state orphenotype of an iPS cell.

One embodiment of the invention is a method for making treated ssRNAsfor use in reprogramming eukaryotic cells that exhibit a firstdifferentiated state or phenotype to cells that exhibit a seconddifferentiated state or phenotype by introducing said ssRNAs into saidcells at least three times over a period of at least three days, saidmethod comprising: (i) treating one or more in vitro-synthesized ssRNAs,each of which encodes a reprogramming factor, with RNase III in areaction mixture comprising divalent magnesium cations at aconcentration of about 1 mM to about 4 mM for sufficient time and underconditions wherein dsRNA is digested to generate treated ssRNAs; and(ii) cleaning up the treated ssRNAs to remove the components of theRNase III reaction mixture and the dsRNA digestion products to generatessRNAs that are at least essentially, practically, extremely orabsolutely free of dsRNA.

In some embodiments, the divalent magnesium cations are at aconcentration of about 1 mM to about 4 mM, or preferably, about 1 mM toabout 3 mM, or more preferably, about 2 mM to about 3 mM, or mostpreferably, about 2 mM.

In some embodiments, the reaction mixture further comprises a monovalentsalt at sufficient concentration wherein the complementary strands ofcontaminant dsRNA remain annealed (e.g., at least about 50 mM,preferably about 50 mM to about 100 mM, more preferably about 100 mM toabout 200 mM, or most preferably about 200 mM). In some embodiments, adivalent salt may be used in place of a monovalent salt, although adivalent salt is not preferred. For example, in some embodiments of themethods, the monovalent salt is selected from the group consisting ofammonium chloride, ammonium acetate, potassium glutamate, potassiumchloride, potassium acetate, sodium acetate, sodium chlorate, lithiumchloride, rubidium chloride and sodium chloride. However, the inventionis not limited to a particular monovalent salt or other salt, althoughsome monovalent salts, such as potassium glutamate and potassiumacetate, are preferred. Any salt that maintains ionic strength so as tomaintain the double-stranded nature of contaminant dsRNA during theRNase III treatment, and in which the RNase III is active and the ssRNAis not degraded, can be used for the method.

In some embodiments, the reaction buffer has a pH in which the invitro-synthesized ssRNA is stable and the RNase III is active (e.g., apH between ˜7 and ˜9).

In accordance with one embodiment, the present invention provides amethod comprising: incubating a dsRNA-specific RNase (e.g., RNase III)with an RNA composition comprising one or more different ssRNA moleculesand contaminant dsRNA molecules, and then cleaning up the ssRNAmolecules in the treated preparation by salt precipitation, PAGE oragarose gel electrophoresis, column chromatography (including using aspin column or HPLC column), or any other methods known in the art,whereby the digested contaminant dsRNA molecules are removed and apurified or treated RNA composition comprising ssRNA molecules isobtained.

In some embodiments, the compositions described above are packaged in akit.

In some of the embodiments of the invention, the method furthercomprises: introducing the purified or treated ssRNAs, wherein saidpurified or treated ssRNAs encode condition-specific (e.g.,cancer-specific) proteins, into human or animal immune cells ex vivo inculture (e.g., T-cells or antigen presenting cells such as dendriticcells) that exhibit a first differentiated state or phenotype (either inculture or in a human or animal subject) and culturing the cells underconditions wherein the cells exhibit a second differentiated state orphenotype wherein they express the condition-specific proteins orpeptides derived therefrom.

In still other embodiments, the purified or treated RNA composition,ssRNAs or mRNAs made using an RNase III treatment method of theinvention, or which comprise a reaction mixture or RNA composition ofthe invention, or which are used in a method for inducing a biologicalor biochemical effect (e.g., for reprogramming) encode one or moretranscription factors, growth factors, cytokines, cluster ofdifferentiation (CD) molecules, interferons, interleukins, cellsignaling proteins, protein receptors, protein hormones, antibodymolecules, or long non-coding RNAs involved in cellular differentiationor maintenance thereof.

In some embodiments, the biological composition comprising RNAcomposition, or ssRNA or mRNA that is substantially, virtually,essentially, practically, extremely or absolutely free of dsRNAmolecules generated using the method comprises or consists of ssRNA ormRNA that encodes a protein on the surface of human cells which isclassified as a cluster of differentiation or cluster of designation(CD) molecule, selected from the group consisting of: CD1a; CD1b; CD1c;CD1d; CD1e; CD2; CD3d; CD3e; CD3g; CD4; CD5; CD6; CD7; CD8a; CD8b; CD9;CD10; CD11a; CD11b; CD11c; CD11d; CDw12; CD14; CD16a; CD16b; CD18; CD19;CD20; CD21; CD22; CD23; CD24; CD25; CD26; CD27; CD28; CD29; CD30; CD31;CD32; CD33; CD34; CD35; CD36; CD37; CD38; CD39; CD40; CD41; CD42a;CD42b; CD42c; CD42d; CD44; CD45; CD46; CD47; CD48; CD49a; CD49b; CD49c;CD49d; CD49e; CD49f; CD50; CD51; CD52; CD53; CD54; CD55; CD56; CD57;CD58; CD59; CD61; CD62E; CD62L; CD62P; CD63; CD64; CD66a; CD66b; CD66c;CD66d; CD66e; CD66f; CD68; CD69; CD70; CD71; CD72; CD74; CD79a; CD79b;CD80; CD81; CD82; CD83; CD84; CD85a; CD85c; CD85d; CD85e; CD85f; CD85g;CD85h; CD85i; CD85j; CD85k; CD86; CD87; CD88; CD89; CD90; CD91; CD92;CD93; CD94; CD95; CD96; CD97; CD98; CD99; CD100; CD101; CD102; CD103;CD104; CD105; CD106; CD107a; CD107b; CD108; CD109; CD110; CD111; CD112;CD113; CD114; CD115; CD116; CD117; CD118; CD119; CD120a; CD120b; CD121a;CD121b; CD122; CD123; CD124; CD125; CD126; CD127; CD129; CD130; CD131;CD132; CD133; CD134; CD135; CD136; CD137; CD138; CD139; CD140a; CD140b;CD141; CD142; CD143; CD144; CD146; CD147; CD148; CD150; CD151; CD152;CD153; CD154; CD155; CD156a; CD156b; CD157; CD158a; CD158b1; CD158b2;CD158c; CD158d; CD158e; CD158f1; CD158g; CD158h; CD158i; CD158j; CD158k;CD158z; CD159a; CD159c; CD160; CD161; CD162; CD163; CD163b; CD164;CD165; CD166; CD167a; CD167b; CD168; CD169; CD170; CD171; CD172a;CD172b; CD172g; CD173; CD177; CD178; CD179a; CD179b; CD180; CD181;CD182; CD183; CD184; CD185; CD186; CD191; CD192; CD193; CD194; CD195;CD196; CD197; CDw198; CDw199; CD200; CD201; CD202b; CD203a; CD203c;CD204; CD205; CD206; CD207; CD208; CD209; CD210; CDw210b; CD212;CD213a1; CD213a2; CD214; CD215; CD217; CD218a; CD218b; CD220; CD221;CD222; CD223; CD224; CD225; CD227; CD228; CD229; CD230; CD231; CD232;CD233; CD234; CD235a; CD235b; CD236; CD238; CD239; CD240CE; CD240D;CD241; CD242; CD243; CD244; CD245; CD246; CD247; CD248; CD249; CD252;CD253; CD254; CD256; CD257; CD258; CD261; CD262; CD263; CD264; CD265;CD266; CD267; CD268; CD269; CD270; CD271; CD272; CD273; CD274; CD275;CD276; CD277; CD278; CD279; CD280; CD281; CD282; CD283; CD284; CD286;CD288; CD289; CD290; CD292; CDw293; CD294; CD295; CD296; CD297; CD298;CD299; CD300a; CD300b; CD300c; CD300d; CD300e; CD300f; CD300g; CD301;CD302; CD303; CD304; CD305; CD306; CD307a; CD307b; CD307c; CD307d;CD307e; CD309; CD312; CD314; CD315; CD316; CD317; CD318; CD319; CD320;CD321; CD322; CD324; CD325; CD326; CD327; CD328; CD329; CD331; CD332;CD333; CD334; CD335; CD336; CD337; CD338; CD339; CD340; CD344; CD349;CD350; CD351; CD352; CD353; CD354; CD355; CD357; CD358; CD360; CD361;CD362; and CD363. In preferred embodiments, the cluster ofdifferentiation molecule is at least practically free, extremely free orabsolutely free of dsRNA molecules.

In some embodiments of the compositions, reaction mixtures, system, kitsand methods of the invention for using any of the foregoing, the invitro-synthesized ssRNA or mRNA encodes a protein selected from thegroup consisting of: erythropoietin (EPO); a detectable enzyme selectedfrom firefly luciferase, Renilla luciferase, bacterialbeta-galactosidase (lacZ), and_green fluorescent protein (GFP); atranscription factor selected from MYC and SRY or MCOP; a growth factoror cytokine selected from the group consisting of platelet-derivedgrowth factor (PDGF), vascular endothelial growth factor (VEGF),transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor(IGF), alpha-melanocyte-stimulating hormone (alpha-MSH); insulin-likegrowth factor-I (IGF-I); IL-4; IL-13; and IL-10; inducible nitric oxidesynthase (iNOS); a heat shock protein; Cystic Fibrosis TransmembraneConductance Regulator (CFTR); an enzyme with antioxidant activityselected from among catalase, phospholipid hydroperoxide glutathioneperoxidase, superoxide dismutase-1, and superoxide dismutase-2; Bruton'styrosine kinase; adenosine deaminase; ecto-nucleoside triphosphatediphosphydrolase; ABCA4; ABCD3; ACADM; AGL; AGT; ALDH4A1; ALPL; AMPD1;APOA2; AVSD1; BRCD2; C1QA; C1QB; C1QG; C8A; C8B; CACNA1S; CCV; CD3Z;CDC2L1; CHML; CHS1; CIAS1; CLCNKB; CMD1A; CMH2; CMM; COL11A1; COL8A2;COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK; DBT; DIO1; DISC1; DPYD; EKV;ENO1; ENO1P; EPB41; EPHX1; F13B; F5; FCGR2A; FCGR2B; FCGR3A; FCHL; FH;FMO3; FMO4; FUCA1; FY; GALE; GBA; GFND; GJA8; GJB3; GLC3B; HF1; HMGCL;HPC1; HRD; HRPT2; HSD3B2; HSPG2; KCNQ4; KCS; KIF1B; LAMB3; LAMC2;LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ; MTHFR; MTR; MUTYH; MYOC; NB; NCF2;NEM1; NPHS2; NPPA; NRAS; NTRK1; OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH;PKLR; PKP1; PLA2G2A; PLOD; PPOX; PPTO; PRCC; PRG4; PSEN2; PTOS1; REN;RFX5; RHD; RMD1; RPE65; SCCD; SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23;SPTA1; TAL1; TNFSF6; TNNT2; TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM;VWS; WS2B; ABCB11; ABCG5; ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP;ALS2; APOB; BDE; BDMR; BJS; BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1;COLAA3; COL4A4; COL6A3; CPS1; CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI; DES;DYSF; EDAR; EFEMP1; EIF2AK3; ERCC3; FSHR; GINGF; GLC1B; GPD2; GYPC;HADHA; HADHB; HOXD13; HPE2; IGKC; IHH; IRS1; ITGA6; KHK; KYNU; LCT;LHCGR; LSFC; MSH2; MSH6; NEB; NMTC; NPHP1; PAFAH1P1; PAX3; PAX8; PMS1;PNKD; PPH1; PROC; REG1A; SAG; SFTPB; SLC11A1; SLC3A1; SOS1; SPG4;SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A@; UV24; WSS; XDH; ZAP70; ZFHX1B;ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET; BBS3; BCHE; BCPM; BTD; CASR; CCR2;CCR5; CDL1; CMT2B; COL7A1; CP; CPO; CRV; CTNNB1; DEM; ETM1; FANCD2; FIH;FOXL2; GBE1; GLB1; GLCLC; GNAI2; GNAT1; GP9; GPX1; HGD; HRG; ITIH1; KNG;LPP; LRS1; MCCC1; MDS1; MHS4; MITF; MLH1; MYL3; MYMY; OPA1; P2RY12;PBXP1; PCCB; POU1F1; PPARG; PROS1; PTHR1; RCA1; RHO; SCA7; SCLC1; SCN5A;SI; SLC25A20; SLC2A2; TF; TGFBR2; THPO; THRB; TKT; TM4SF1; TRH; UMPS;UQCRC1; USH3A; VHL; WS2A; XPC; ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2; ALB;ASMD; BFHD; CNGA1; CRBM; DCK; DSPP; DTDP2; ELONG; ENAM; ETFDH; EVC; F11;FABP2; FGA; FGB; FGFR3; FGG; FSHMD1A; GC; GNPTA; GNRHR; GYPA; HCA; HCL2;HD; HTN3; HVBS6; IDUA; IF; JPD; KIT; KLKB1; LQT4; MANBA; MLLT2; MSX1;MTP; NR3C2; PBT; PDE6B; PEE1; PITX2; PKD2; QDPR; SGCB; SLC25A4; SNCA;SOD3; STATH; TAPVR1; TYS; WBS2; WFS1; WHCR; ADAMTS2; ADRB2; AMCN; AP3B1;APC; ARSB; B4GALT7; BHR1; C6; C7; CCAL2; CKN1; CMDJ; CRHBP; CSF1R; DHFR;DIAPH1; DTR; EOS; EPD; ERVR; F12; FBN2; GDNF; GHR; GLRA1; GM2A; HEXB;HSD17B4; ITGA2; KFS; LGMDLA; LOX; LTC4S; MAN2A1; MCC; MCCC2; MSH3; MSX2;NR3C1; PCSK1; PDE6A; PFBI; RASA1; SCZD1; SDHA; SGCD; SLC22A5; SLC26A2;SLC6A3; SM1; SMA@; SMN1; SMN2; SPINK5; TCOF1; TELAB1; TGFBI; ALDH5A1;ARG1; AS; ASSP2; BCKDHB; BF; C2; C4A; CDKN1A; COL10A1; COL11A2; CYP21A2;DYX2; EJM1; ELOVL4; EPM2A; ESR1; EYA4; F13A1; FANCE; GCLC; GJA1; GLYS1;GMPR; GSE; HCR; HFE; HLA-A; HLA-DPB1; HLA-DRA; HPFH; ICS1; IDDM1;IFNGR1; IGAD1; IGF2R; ISCW; LAMA2; LAP; LCA5; LPA; MCDR1; MOCS1; MUT;MYB; NEU1; NKS1; NYS2; OA3; ODDD; OFCO; PARK2; PBCA; PBCRA1; PDB1; PEX3;PEX6; PEX7; PKHD1; PLA2G7; PLG; POLH; PPAC; PSORS1; PUJO; RCD1; RDS;RHAG; RP14; RUNX2; RWS; SCA1; SCZD3; SIASD; SOD2; ST8; TAP1; TAP2;TFAP2B; TNDM; TNF; TPBG; TPMT; TULP1; WISP3; AASS; ABCB1; ABCB4; ACHE;AQP1; ASL; ASNS; AUTS1; BPGM; BRAF; C7orf2; CACNA2D1; CCM1; CD36; CFTR;CHORDOMA; CLCN1; CMH6; CMT2D; COL1A2; CRS; CYMD; DFNA5; DLD; DYT11;EEC1; ELN; ETV1; FKBP6; GCK; GHRHR; GHS; GLI3; GPDS1; GUSB; HLXB9;HOXA13; HPFH2; HRX; IAB; IMMP2L; KCNH2; LAMBI; LEP; MET; NCF1; NM; OGDH;OPN1SW; PEX1; PGAM2; PMS2; PON1; PPP1R3A; PRSS1; PTC; PTPN12; RP10; RP9;SERPINE1; SGCE; SHFM1; SHH; SLC26A3; SLC26A4; SLOS; SMAD1; TBXAS1;TWIST; ZWS1; ACHM3; ADRB3; ANK1; CA1; CA2; CCAL1; CLN8; CMT4A; CNGB3;COH1; CPP; CRH; CYPIIB1; CYPIIB2; DECR1; DPYS; DURS1; EBS1; ECA1; EGI;EXT1; EYA1; FGFR1; GNRH1; GSR; GULOP; HR; KCNQ3; KFM; KWE; LGCR; LPL;MCPH1; MOS; MYC; NAT1; NAT2; NBS1; PLAT; PLEC1; PRKDC; PXMP3; RP1;SCZD6; SFTPC; SGM1; SPG5A; STAR; TG; TRPS1; TTPA; VMD1; WRN; ABCA1;ABL1; ABO; ADAMTS13; AK1; ALAD; ALDH1A1; ALDOB; AMBP; AMCD1; ASS; BDMF;BSCL; C5; CDKN2A; CHAC; CLA1; CMD1B; COL5A1; CRAT; DBH; DNAI1; DYS;DYT1; ENG; FANCC; FBP1; FCMD; FRDA; GALT; GLDC; GNE; GSM1; GSN; HSD17B3;HSN1; IBM2; INVS; JBTS1; LALL; LCCS1; LCCS; LGMD2H; LMX1B; MLLT3; MROS;MSSE; NOTCH1; ORM1; PAPPA; PIP5KIB; PTCH; PTGS1; RLN1; RLN2; RMRP; ROR2;RPD1; SARDH; SPTLC1; STOM; TDFA; TEK; TMC1; TRIM32; TSC1; TYRP1; XPA;CACNB2; COL17A1; CUBN; CXCL12; CYP17; CYP2C19; CYP2C9; EGR2; EMX2;ERCC6; FGFR2; HK1; HPS1; IL2RA; LGI1; LIPA; MAT1A; MBL2; MKI67; MXI1;NODAL; OAT; OATL3; PAX2; PCBD; PEO1; PHYH; PNLIP; PSAP; PTEN; RBP4;RDPA; RET; SFTPA1; SFTPD; SHFM3; SIAL; THC2; TLX1; TNFRSF6; UFS; UROS;AA; ABCC8; ACAT1; ALX4; AMPD3; ANC; APOAL; APOA4; APOC3; ATM; BSCL2;BWS; CALCA; CAT; CCND1; CD3E; CD3G; CD59; CDKNLC; CLN2; CNTF; CPT1A;CTSC; DDB1; DDB2; DHCR7; DLAT; DRD4; ECB2; ED4; EVR1; EXT2; F2; FSHB;FTH1; G6PT1; G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1; HBG2; HMBS; HND;HOMG2; HRAS; HVBS1; IDDM2; IGER; INS; JBS; KCNJ11; KCNJ1; KCNQ1; LDHA;LRP5; MEN1; MLL; MYBPC3; MYO7A; NNO1; OPPG; OPTB1; PAX6; PC; PDX1; PGL2;PGR; PORC; PTH; PTS; PVRL1; PYGM; RAG1; RAG2; ROM1; RRAS2; SAA1; SCA5;SCZD2; SDHD; SERPING1; SMPD1; TCIRG1; TCL2; TECTA; TH; TREH; TSG101;TYR; USH1C; VMD2; VRNI; WT1; WT2; ZNF145; A2M; AAAS; ACADS; ACLS;ACVRL1; ALDH2; AMHR2; AOM; AQP2; ATD; ATP2A2; BDC; CIR; CD4; CDK4; CNA1;COL2A1; CYP27B1; DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1;HMGA2; HMN2; HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A; KRT3; KRT4;KRT5; KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL2; OAP; PAH;PPKB; PRB3; PTPN11; PXR1; RLS; RSN; SAS; SAX1; SCA2; SCNN1A; SMAL; SPPM;SPSMA; TBX3; TBX5; TCF1; TPI1; TSC3; ULR; VDR; VWF; ATP7B; BRCA2; BRCD1;CLN5; CPB2; ED2; EDNRB; ENUR1; ERCC5; F10; F7; GJB2; GJB6; IPF1; MBS1;MCOR; NYS4; PCCA; RB1; RHOK; SCZD7; SGCG; SLC10A2; SLC25A15; STARP1;ZNF198; ACHM1; ARVD1; BCH; CTAA1; DAD1; DFNB5; EML1; GALC; GCH1; IBGC1;IGH@; IGHC group; IGHG1; IGHM; IGHR; IV; LTBP2; MJD; MNG1; MPD1; MPS3C;MYH6; MYH7; NP; NPC2; PABPN1; PSEN1; PYGL; RPGRIP1; SERPINA1; SERPINA3;SERPINA6; SLC7A7; SPG3A; SPTB; TCL1A; TGM1; TITF1; TMIP; TRA@; TSHR;USHLA; VP; ACCPN; AHO2; ANCR; B2M; BBS4; BLM; CAPN3; CDAN1; CDAN3; CLN6;CMH3; CYP19; CYP1A1; CYP1A2; DYX1; EPB42; ETFA; EYCL3; FAH; FBN1; FES;HCVS; HEXA; IVD; LCS1; LIPC; MYO5A; OCA2; OTSC1; PWCR; RLBP1; SLC12A1;SPG6; TPM1; UBE3A; WMS; ABCC6; ALDOA; APRT; ATP2A1; BBS2; CARD15; CATM;CDH1; CETP; CHST6; CLN3; CREBBP; CTH; CTM; CYBA; CYLD; DHS; DNASE1;DPEP1; ERCC4; FANCA; GALNS; GAN; HAGH; HBA1; HBA2; HBHR; HBQ1; HBZ;HBZP; HP; HSD11B2; IL4R; LIPB; MC1R; MEFV; MHC2TA; MLYCD; MMVP1; PHKB;PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1; SCA4; SCNN1B; SCNN1G; SLC12A3;TAT; TSC2; VDI; WT3; ABR; ACACA; ACADVL; ACE; ALDH3A2; APOH; ASPA;AXIN2; BCL5; BHD; BLMH; BRCA1; CACD; CCA1; CCZS; CHRNB1; CHRNE; CMT1A;COL1A1; CORD5; CTNS; EPX; ERBB2; G6PC; GAA; GALK1; GCGR; GFAP; GH1; GH2;GPIBA; GPSC; GUCY2D; ITGA2B; ITGB3; ITGB4; KRT10; KRT12; KRT13; KRT14;KRT14L1; KRT14L2; KRT14L3; KRT16; KRT16L1; KRT16L2; KRT17; KRT9; MAPT;MDB; MDCR; MGI; MHS2; MKS1; MPO; MYO15A; NAGLU; NAPB; NF1; NME1; P4HB;PAFAH1B1; PECAM1; PEX12; PHB; PMP22; PRKAR1A; PRKCA; PRKWNK4; PRP8;PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A; SERPINF2; SGCA; SGSH;SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9; SSTR2; SYM1; SYNS1;TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH; ATP8B1; BCL2; CNSN;CORD1; CYB5; DCC; F5F8D; FECH; FEO; LAMA3; LCFS2; MADH4; MAFD1; MC2R;MCL; MYP2; NPC1; SPPK; TGFBRE; TGIF; TTR; AD2; AMH; APOC2; APOE; ATHS;BAX; BCKDHA; BCL3; BFIC; C3; CACNA1A; CCO; CEACAM5; COMP; CRX; DBA; DDU;DFNA4; DLL3; DM1; DMWD; E11S; ELA2; EPOR; ERCC2; ETFB; EXT3; EYCL1; FTL;FUT1; FUT2; FUT6; GAMT; GCDH; GPI; GUSM; HB1; HCL1; HHC2; HHC3; ICAM3;INSR; JAK3; KLK3; LDLR; LHB; LIG1; LOH19CR1; LYL1; MAN2B1; MCOLN1; MDRV;MLLT1; NOTCH3; NPHS1; OFC3; OPA3; PEPD; PRPF31; PRTN3; PRX; PSG1; PVR;RYR1; SLC5A5; SLC7A9; STK11; TBXA2R; TGFB1; TNNI3; TYROBP; ADA; AHCY;AVP; CDAN2; CDPD1; CHED1; CHED2; CHRNA4; CST3; EDN3; EEGV1; FTLL1; GDF5;GNAS; GSS; HNF4A; JAG1; KCNQ2; MKKS; NBIA1; PCK1; PI3; PPCD; PPGB; PRNP;THBD; TOP1; AIRE; APP; CBS; COL6A1; COL6A2; CSTB; DCR; DSCR1; FPDMM;HLCS; HPE1; ITGB2; KCNE1; KNO; PRSS7; RUNX1; SOD1; TAM; ADSL; ARSA; BCR;CECR; CHEK2; COMT; CRYBB2; CSF2RB; CTHM; CYP2D6; CYP2D7P1; DGCR; DIA1;EWSR1; GGT1; MGCR; MN1; NAGA; NE2; OGS2; PDGFB; PPARA; PRODH; SCO2;SCZD4; SERPIND1; SLC5A1; SOX10; TCN2; TIMP3; TST; VCF; ABCD1; ACTL1;ADFN; AGMX2; AHDS; AIC; AIED; AIH3; ALAS2; AMCD; AMELX; ANOP1; AR;ARAF1; ARSC2; ARSE; ARTS; ARX; ASAT; ASSP5; ATP7A; ATRX; AVPR2; BFLS;BGN; BTK; BZX; C1HR; CACNA1F; CALB3; CBBM; CCT; CDR1; CFNS; CGF1; CHM;CHR39c; CIDX; CLA2; CLCN5; CLS; CMTX2; CMTX3; CND; COD1; COD2; COL4A5;COL4A6; CPX; CVD1; CYBB; DCX; DFN2; DFN4; DFN6; DHOF; DIAPH2; DKC1; DMD;DSS; DYT3; EBM; EBP; ED1; ELK1; EMD; EVR2; F8; F9; FCP1; FDPSL5; FGD1;FGS1; FMR1; FMR2; G6PD; GABRA3; GATA1; GDI1; GDXY; GJB1; GK; GLA; GPC3;GRPR; GTD; GUST; HMS1; HPRT1; HPT; HTC2; HTR2c; HYR; IDS; IHG1; IL2RG;INDX; IP1; IP2; JMS; KAL1; KFSD; LiCAM; LAMP2; MAA; MAFD2; MAOA; MAOB;MCF2; MCS; MEAX; MECP2; MF4; MGC1; MIC5; MID1; MLLT7; MLS; MRSD; MRX14;MRX1; MRX20; MRX2; MRX3; MRX40; MRXA; MSD; MTM1; MYCL2; MYP1; NDP; NHS;NPHL1; NROB1; NSX; NYS1; NYX; OA1; OASD; OCRL; ODT1; OFD1; OPA2; OPD1;OPEM; OPNlLW; OPNIMW; OTC; P3; PDHA1; PDR; PFC; PFKFB1; PGK1; PGK1P1;PGS; PHEX; PHKA1; PHKA2; PHP; PIGA; PLP1; POF1; POLA; POU3F4; PPMX; PRD;PRPS1; PRPS2; PRS; RCCP2; RENBP; RENS1; RP2; RP6; RPGR; RPS4X; RPS6KA3;RS1; S11; SDYS; SEDL; SERPINA7; SH2D1A; SHFM2; SLC25A5; SMAX2; SRPX;SRS; STS; SYN1; SYP; TAF1; TAZ; TBX22; TDD; TFE3; THAS; THC; TIMM8A;TIM1; TKCR; TNFSF5; UBE1; UBE2A; WAS; WSN; WTS; WWS; XIC; XIST; XK; XM;XS; ZFX; ZIC3; ZNF261; ZNF41; ZNF6; AMELY; ASSP6; AZF1; AZF2; DAZ; GCY;RPS4Y; SMCY; ZFY; ABAT; AEZ; AFA; AFD1; ASAH1; ASD1; ASMT; CCAT; CECR9;CEPA; CLA3; CLN4; CSF2RA; CTS1; DF; DIH1; DWS; DYT2; DYT4; EBR3; ECT;EEF1A1L14; EYCL2; FANCB; GCSH; GCSL; GIP; GTS; HHG; HMI; HOAC; HOKPP2;HRPT1; HSD3B3; HTC1; HV1S; ICHQ; ICR1; ICR5; IL3RA; KAL2; KMS; KRT18;KSS; LCAT; LHON; LIMM; MANBB; MCPH2; MEB; MELAS; MIC2; MPFD; MS; MSS;MTATP6; MTCO1; MTC03; MTCYB; MTND1; MTND2; MTND4; MTND5; MTND6; MTRNR1;MTRNR2; MTTE; MTTG; MTTI; MTTK MTTL1; MTTL2; MTTN; MTTP; MTTS1; NAMSD;OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1; RBS;RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; and TD.

In some embodiments of any of the compositions, methods, and systems forinducing a biological or biochemical effect by repeatedly orcontinuously introducing a ssRNA or mRNA into a mammalian cell (e.g.,that exhibits a first state of differentiation or phenotype, e.g., forreprogramming to a second state of differentiation or phenotype), themammalian cell is selected from the group consisting of: anantigen-presenting cell, a dendritic cell, a macrophage, a neural cell,a brain cell, an astrocyte, a microglial cell, and a neuron, a spleencell, a lymphoid cell, a lung cell, a lung epithelial cell, a skin cell,a keratinocyte, an endothelial cell, an alveolar cell, an alveolarmacrophage, an alveolar pneumocyte, a vascular endothelial cell, amesenchymal cell, an epithelial cell, a colonic epithelial cell, ahematopoietic cell, a bone marrow cell, a Claudius' cell, Hensen cell,Merkel cell, Muller cell, Paneth cell, Purkinje cell, Schwann cell,Sertoli cell, acidophil cell, acinar cell, adipoblast, adipocyte, brownor white alpha cell, amacrine cell, beta cell, capsular cell,cementocyte, chief cell, chondroblast, chondrocyte, chromaffin cell,chromophobic cell, corticotroph, delta cell, Langerhans cell, folliculardendritic cell, enterochromaffin cell, ependymocyte, epithelial cell,basal cell, squamous cell, endothelial cell, transitional cell,erythroblast, erythrocyte, fibroblast, fibrocyte, follicular cell, germcell, gamete, ovum, spermatozoon, oocyte, primary oocyte, secondaryoocyte, spermatid, spermatocyte, primary spermatocyte, secondaryspermatocyte, germinal epithelium, giant cell, glial cell, astroblast,astrocyte, oligodendroblast, oligodendrocyte, glioblast, goblet cell,gonadotroph, granulosa cell, haemocytoblast, hair cell, hepatoblast,hepatocyte, hyalocyte, interstitial cell, juxtaglomerular cell,keratinocyte, keratocyte, lemmal cell, leukocyte, granulocyte, basophil,eosinophil, neutrophil, lymphoblast, B-lymphoblast, T-lymphoblast,lymphocyte, B-lymphocyte, T-lymphocyte, helper induced T-lymphocyte, Th1T-lymphocyte, Th2 T-lymphocyte, natural killer cell, thymocyte,macrophage, Kupffer cell, alveolar macrophage, foam cell, histiocyte,luteal cell, lymphocytic stem cell, lymphoid cell, lymphoid stem cell,macroglial cell, mammotroph, mast cell, medulloblast, megakaryoblast,megakaryocyte, melanoblast, melanocyte, mesangial cell, mesothelialcell, metamyelocyte, monoblast, monocyte, mucous neck cell, myoblast,myocyte, muscle cell, cardiac muscle cell, skeletal muscle cell, smoothmuscle cell, myelocyte, myeloid cell, myeloid stem cell, myoblast,myoepithelial cell, myofibrobast, neuroblast, neuroepithelial cell,neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell,parafollicular cell, paraluteal cell, peptic cell, pericyte, peripheralblood mononuclear cell, phaeochromocyte, phalangeal cell, pinealocyte,pituicyte, plasma cell, platelet, podocyte, proerythroblast,promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte,retinal pigment epithelial cell, retinoblast, small cell, somatotroph,stem cell, sustentacular cell, teloglial cell, and a zymogenic cell.

In some embodiments of all of the methods, the purified or treated RNAcomposition does not generate an innate immune response that issufficient to cause significant inhibition of cellular protein synthesisor dsRNA-induced apoptosis. In certain embodiments, the purified ortreated RNA composition does not generate an innate immune response thatis sufficient to cause significant inhibition of cellular proteinsynthesis or dsRNA-induced apoptosis when said introducing of thepurified RNA composition into a living human or animal cell or subjectis repeated at least 3 times (e.g., when introduced daily for multipleweeks or daily or weekly for multiple weeks, months or years). Inpreferred embodiments of the method for reprogramming a human or animalsomatic cell to an iPS cell, the purified or treated RNA compositiondoes not generate an innate immune response that is sufficient to causesubstantial inhibition of cellular protein synthesis or dsRNA-inducedapoptosis when said introducing of the purified or treated RNAcomposition into a living human or animal cell is repeated daily forabout 10-18 or more days.

In some embodiments, the purified or treated ssRNAs are introduced dailyor twice per day, with said introducing occurring about 1 time per week,2 times per week, 3 times per week, 4 times per week, 5 times per week,6 times per week, or daily for a period consisting of: (i) up to about 4weeks for cells in culture; or (ii) for a period of weeks, months oryears for the living human or animal subject.

In certain embodiments, the invention provides a method for treating,reducing or eliminating a symptom or disease of a human or animalsubject that exhibits a disease condition, comprising: administering tothe human or animal subject an effective dose of purified or treatedssRNAs, whereby the symptom or disease is reduced or eliminated.

In some embodiments the treated ssRNA or the purified or treated ssRNAis used to: reprogram cells that exhibit a first differentiated state orphenotype to cells that exhibit a second differentiated state orphenotype; compensate for a missing or defective protein; express adesired protein such as a transcription factor, cell signaling protein,growth factor, interferon, interleukin, cluster of differentiation (CD)molecule (e.g., see http://www. followed by“uniprot.org/docs/cdlist.txt”), protein hormone, protein receptor, or anantibody; express a long non-coding RNA molecule involved withdifferentiation (e.g., “HOTAIR” OR HOX antisense intergenic RNA; Wan Yand Chang H Y, 2010); or modulate or trigger a disease-specific immuneresponse.

In certain embodiments, the invention provides a method forreprogramming a eukaryotic cell that exhibits a first differentiatedstate or phenotype to a cell that exhibits a second differentiated stateor phenotype. Thus, in certain embodiments, the method furthercomprises: introducing the treated ssRNAs or the purified ssRNAs into ahuman or animal cell that exhibits a first differentiated state orphenotype and culturing the cell under conditions wherein the cellexhibits a second differentiated state or phenotype. In one preferredembodiment of this method, the treated ssRNAs or the purified ssRNAs arepurified ssRNAs that encode a protein. In one preferred embodiment ofthis method, the purified ssRNAs encode induced pluripotent stem cell(iPSC) induction factors, the cells that exhibit a first differentiatedstate are human or animal somatic cells, and the purified ssRNAs areintroduced into said cells daily for about 7 to about 21 days togenerate cells that exhibit a second differentiated state or phenotypecomprising iPSCs.

In certain embodiments, the invention provides a method for reducing oreliminating a symptom or disease of a human or animal subject thatexhibits a disease condition, comprising: administering introducing intothe subject the cell that exhibits the second differentiated state orphenotype, whereby the symptom or disease is reduced or eliminated.

In some preferred embodiments of the methods, the one or more invitro-synthesized ssRNAs and/or the purified ssRNAs exhibit at least oneheterologous 5′ UTR sequence, Kozak sequence, IRES sequence, or 3′ UTRsequence that results in greater translation into the encoded proteinwhen said respective ssRNAs are introduced into eukaryotic cellscompared to the same ssRNAs that do not exhibit said respective 5′ UTRsequence, Kozak sequence, IRES sequence, or 3′ UTR sequence. In someparticular preferred embodiments, the 5′ UTR or 3′ UTR is a sequenceexhibited by a Xenopus or human alpha- (α-) globin or beta- (β-) globinmRNA, or wherein the 5′ UTR is a sequence exhibited by tobacco etchvirus (TEV) RNA.

In some embodiments of the methods, the treated ssRNAs or the purifiedssRNAs exhibit a 5′ cap comprising 7-methylguanine or an anti-reversecap analog (ARCA). In some embodiments, the treated ssRNAs or thepurified ssRNAs further comprise a 5′ cap that has a cap1 structure,wherein the 2′ hydroxyl of the ribose in the 5′ penultimate nucleotideis methylated (e.g., using RNA 2′-O-methyltransferase, e.g., using theSCRIPTCAP™ 2′-O-methyltransferase kit, CELLSCRIPT, Inc.).

In some embodiments, wherein the treated ssRNAs or the purified ssRNAsexhibit a 5′ cap, the one or more in vitro-synthesized ssRNAs used forsaid treating in said method exhibit the 5′ cap (i.e., prior to saidtreating). Thus, in some embodiments, the one or more invitro-synthesized ssRNAs used for said treating comprise capped ssRNAs.In some of these embodiments, the one or more in vitro-synthesized ssRNAmolecules that exhibit the 5′ cap were synthesized prior to their usefor said treating: (i) co-transcriptionally by incorporation of a capanalog (e.g., an anti-reverse cap analog or ARCA) during in vitrotranscription of (e.g., using the MESSAGEMAX™ T7 ARCA-capped messagetranscription kit or the INCOGNITO™ T7 ARCA 5^(m)C- and Ψ-RNAtranscription kit, CELLSCRIPT, Inc., Madison, Wis., USA); or (ii)post-transcriptionally by incubating in vitro-transcribed ssRNAmolecules with a capping enzyme system comprising RNA guanyltransferaseunder conditions wherein the in vitro-transcribed ssRNA molecules are5′-capped, including wherein the capping enzyme system results inmethylation of the 2′ hydroxyl of the ribose in the 5′ penultimatenucleotide (e.g., using T7 mSCRIPT™ standard mRNA production system, orusing a separate in vitro transcription system, such as the T7-SCRIBE™standard RNA IVT kit, the INCOGNITO™ T7 Ψ-RNA transcription kit, or theINCOGNITO™ T7 5mC- and Ψ-RNA transcription kit to obtain ssRNA, and theSCRIPTCAP™ m⁷G capping system to obtain cap0 RNA (all from CELLSCRIPT,Inc.); in some embodiments, the capping enzyme system further results inmethylation of the 2′ hydroxyl of the ribose in the 5′ penultimatenucleotide to generate cap1 RNA, and the method further comprises:incubating with RNA 2′-O-methyltransferase (e.g., using the SCRIPTCAP™2′-O-methyltransferase kit, CELLSCRIPT, Inc.).

In some preferred embodiments wherein the treated ssRNAs or the purifiedssRNAs exhibit a 5′ cap, the one or more in vitro-synthesized ssRNAsused in said method for said treating are uncapped and the methodfurther comprises: post-transcriptionally capping the treated ssRNAs orthe purified ssRNAs to generate 5′ capped treated ssRNAs or 5′ cappedpurified ssRNAs. In some embodiments, said post-transcriptional cappingof the treated ssRNAs or the purified ssRNAs is performed as describedabove and/or in the product literature provided with the SCRIPTCAP™ m⁷GCapping System, the SCRIPTCAP™ 2′-O-methyltransferase kit, or the 17mSCRIPT™ standard mRNA production system with respect to the cappingenzyme system components (all from CELLSCRIPT, Inc., Madison, Wis.,USA).

In some preferred embodiments, the one or more in vitro-synthesizedssRNAs used for said treating are substantially free of uncapped RNAsthat exhibit a 5′-triphosphate group (which are considered to be onetype of “contaminant RNA molecules” herein). In some preferredembodiments, the treated ssRNAs and/or the purified ssRNAs generatedfrom a method are substantially free of uncapped RNAs that exhibit a5′-triphosphate group. In certain embodiments, the one or more invitro-synthesized ssRNAs used for said treating, the treated ssRNAs,and/or the purified ssRNAs consist of a population of ssRNA moleculeshaving: (i) greater than 90% capped ssRNA molecules; (ii) greater than95% capped ssRNA molecules; (iii) greater than 99% capped ssRNAmolecules; or (iv) greater than 99.9% capped ssRNA molecules. In someembodiments wherein the population of ssRNA molecules also comprisescontaminant uncapped RNA molecules that exhibit a 5′-triphosphate group,the method further comprises: incubating the one or more invitro-synthesized ssRNAs used for said treating, or the treated ssRNAsor the purified ssRNAs generated from the method with an alkalinephosphatase (e.g., NTPhosphatase™, epicentre technologies, Madison,Wis., USA) or with RNA 5′ polyphosphatase (epicentre technologies) toremove the triphosphate groups from contaminating uncapped ssRNAs; insome embodiments, the one or more in vitro-synthesized ssRNAs used forsaid treating, or the treated ssRNAs or the purified ssRNAs that areincubated with RNA 5′polyphosphatase are further incubated withTERMINATOR™ 5′-phosphate-dependent nuclease or Xm1 exoribonuclease(e.g., from Saccharomyces cerevisae) to digest said contaminatinguncapped ssRNAs. These methods for incubating with alkaline phosphataseor with RNA 5′ polyphosphatase and TERMINATOR™ 5′-phosphate-dependentnuclease or Xm1 exoribonuclease are particularly useful to removeuncapped ssRNAs from capped ssRNAs that were made by co-transcriptionalcapping by incorporating a cap analog during an in vitro transcriptionreaction.

In some preferred embodiments of the methods, the one or more invitro-synthesized ssRNAs, the treated ssRNAS, or the purified ssRNAs arepolyadenylated. In some embodiments, the one or more invitro-synthesized ssRNAs, the treated ssRNAS, or the purified ssRNAsexhibit a poly-A tail of about 50 to about 200 nucleotides. However thepoly-A tail is not limited with respect to the number of nucleotides andthe poly-A tail can exhibit more than 200 or less than 50 nucleotides.

In some embodiments, the one or more in vitro-synthesized ssRNAs, thetreated ssRNAS, and/or the purified ssRNAs exhibit a poly-A tail of50-100 nucleotides, 100-200 nucleotides, 150-200 nucleotides, or greaterthan 200 nucleotides. In some preferred embodiments, the one or more invitro-synthesized ssRNAs, the treated ssRNAS, and/or the purified ssRNAsexhibit a poly-A tail of 150-200 nucleotides in length. In someembodiments, the one or more in vitro-synthesized ssRNAs arepolyadenylated by in vitro transcription of a DNA template thatcomprises a terminal oligo(dT) sequence that is complementary to thepoly-A tail. In some preferred embodiments, the one or more invitro-synthesized ssRNAs, the treated ssRNAS, or the purified ssRNAs arepolyadenylated by post-transcriptional polyadenylation using a poly(A)polymerase or poly-A polymerase (e.g., poly-A polymerase derived from E.coli or Saccharomyces cerevisiae; or a poly-A polymerase from acommercial source, e.g., A-PLUS™ poly(A) polymerase, CELLSCRIPT, Inc.,Madison, Wis. 53713, USA). However, unless specifically stated withrespect to a particular method, the invention is not limited to use of aparticular poly(A) polymerase, and any suitable poly(A) polymerase canbe used. A “poly(A) polymerase” or “poly-A polymerase” or “PAP”, whenused herein, means a template-independent RNA polymerase found in mosteukaryotes, prokaryotes, and eukaryotic viruses that selectively usesATP to incorporate AMP residues to 3′-hydroxylated ends of RNA. SincePAP enzymes that have been studied from plants, animals, bacteria andviruses all catalyze the same overall reaction (e.g., see Edmonds, M,1990), are highly conserved structurally (e.g., see Gershon, P, 2000),and lack intrinsic specificity for particular sequences or sizes of RNAmolecules if the PAP is separated from proteins that recognize AAUAAApolyadenylation signals (Wilusz, J and Shenk, T, 1988), purifiedwild-type and recombinant PAP enzymes from any of a variety of sourcescan be used in the kits and methods of the present invention. Theinvention is also not limited to the methods for polyadenylating the oneor more in vitro-synthesized ssRNAs, the treated ssRNAS, or the purifiedssRNAs described herein and any other suitable method in the art may beused for said polyadenylating.

In some embodiments of the methods, the one or more in vitro-synthesizedssRNAs comprise at least one modified ribonucleoside selected from thegroup consisting of pseudouridine (Ψ), 1-methyl-pseudouridine (m¹Ψ),5-methylcytidine (m⁵C), 5-methyluridine (m⁵U), 2′-O-methyluridine (Um orm^(2′-O)U), 2-thiouridine (s²U), and N⁶-methyladenosine (m⁶A) in placeof at least a portion of the corresponding unmodified canonicalribonucleoside. In some embodiments wherein the one or more invitro-synthesized ssRNAs comprise at least one modified ribonucleoside,the at least one modified ribonucleoside is selected from the groupconsisting of: (i) pseudouridine (Ψ), 1-methyl-pseudouridine (m¹Ψ),5-methyluridine (m⁵U), 2′-O-methyluridine (Um or m^(2′-O)U), and2-thiouridine (s²U) in place of all or substantially all of thecanonical uridine residues; (ii) 5-methylcytidine (m⁵C) in place of allor substantially all of the canonical cytidine residues; and/or (iii)N⁶-methyladenosine (m⁶A) in place of all or substantially all of thecanonical adenosine residues. In some preferred embodiments wherein theone or more in vitro-synthesized ssRNAs comprise at least one modifiedribonucleoside, the at least one modified ribonucleoside consists ofpseudouridine (Ψ) or 1-methyl-pseudouridine (m¹Ψ) in place of all orsubstantially all of the canonical uridine residues, and/or5-methylcytidine (m⁵C) in place of all or substantially all of thecanonical cytidine residues. In some preferred embodiments, wherein thein vitro-synthesized ssRNAs comprise pseudouridine (Ψ) or1-methyl-pseudouridine (m¹Ψ) in place of all or substantially all of thecanonical uridine residues, the in vitro-synthesized ssRNAs alsocomprise 5-methylcytidine (m⁵C) in place of all or substantially all ofthe canonical cytidine residues.

In some embodiments of the methods wherein the one or more invitro-synthesized ssRNAs comprise at least one modified ribonucleoside,the one or more in vitro-synthesized ssRNAs are synthesized by in vitrotranscription (IVT) of a DNA template that encodes each said at leastone protein or polypeptide reprogramming factor using an RNA polymerasethat initiates said transcription from a cognate RNA polymerase promoterthat is joined to said DNA template and ribonucleoside 5′ triphosphates(NTPs) comprising at least one modified ribonucleoside 5′ triphosphateselected from the group consisting of pseudouridine 5′ is triphosphate(ΨTP), 1-methyl-pseudouridine 5′ triphosphate (m¹ΨP), 5-methylcytidine5′ triphosphate (m⁵CTP), 5-methyluridine 5′ triphosphate (m⁵UTP),2′-O-methyluridine 5′ triphosphate (UmTP or m^(2′-O)UTP), 2-thiouridine5′ triphosphate (s²UTP), and N⁶-methyladenosine 5′ triphosphate (m⁶ATP);in some preferred embodiments, the modified NTP is used in place of allor substantially all of the corresponding unmodified NTP in the IVTreaction (e.g., ωTP, m¹ΨP, m⁵UTP, m^(2′-O)UTP or s²UTP in place of UTP:m⁵CTP in place of CTP; or m⁶ATP in place of ATP).

In some embodiments of the methods, the one or more in vitro-synthesizedssRNAs are substantially free of modified ribonucleosides (other thanthose ribonucleosides comprising the 5′ cap structure, if a 5′ cap ispresent, including the 5′ penultimate nucleoside when the one or more invitro-synthesized ssRNAs exhibit a cap1 cap structure). In someembodiments of the methods, except for the ribonucleosides comprisingthe 5′ cap, if present, the one or more in vitro-synthesized ssRNAscomprise only the canonical ribonucleosides G, A, C and U. In someembodiments of the methods, the one or more in vitro-synthesized ssRNAsthat encode each said at least one protein or polypeptide reprogrammingfactor was synthesized by in vitro transcription of a DNA template by anRNA polymerase using the canonical NTPs: GTP, ATP, CTP and UTP.

In some of the embodiments of the method for making purified ssRNAswherein the one or more in vitro-synthesized ssRNAs comprise either oneor more modified ribonucleosides (e.g. Ψ and/or m⁵C) or only unmodifiedribonucleosides (G, A, C and U) and encode one or more protein orpolypeptide reprogramming factors, the method further comprises:introducing the purified or treated ssRNAs into a eukaryotic cell thatexhibits a first differentiated state or phenotype at least three timesover a period of at least three days and culturing the cells underconditions wherein the cells exhibit a second differentiated state orphenotype. In some of these embodiments, the eukaryotic cell thatexhibits a first differentiated state or phenotype is a human or animalsomatic cell, the purified or treated ssRNAs encode reprogrammingfactors comprising induced pluripotent stem cell (iPS cell) inductionfactors, and the cells that exhibit a second differentiated state orphenotype are iPS cells; in these embodiments the introducing of thepurified or treated ssRNAs at least three times over a period of atleast three days means about at least seven times over at least sevendays to about at least 21 times over at least 21 days.

Surprisingly and unexpectedly, the present Applicants found that thismethod for reprogramming eukaryotic cells by introducing into the cellspurified or treated ssRNAs encoding iPS cell induction factors resultedin reprogramming of human or animal somatic cells (e.g., fibroblasts,kerotinocytes) to iPS cells, both when purified or treated ssRNAscomprising modified ribonucleosides such as Ψ and/or m⁵C were used, andwhen purified or treated ssRNAs consisting of only unmodified canonicalribonucleosides, G, A, C and U, were used. Prior to the results of thepresent Applicants, it is believed that only modified ssRNAs had beenused for reprogramming cells. Prior to the results of the presentApplicants, it is believed that nobody had ever shown reprogramming ofhuman or animal somatic cells to iPS cells with ssRNAs consisting ofonly unmodified canonical ribonucleosides.

Still further, prior to the work disclosed in the present application,it is believed that nobody had ever demonstrated reprogramming of ahuman or animal somatic cell to an iPS cell using modified ssRNAswithout contacting the cells with an inhibitor of the interferonsignaling pathway, such as the B18R protein as an inhibitor of type Iinterferon, prior to introducing said ssRNAs encoding the iPS cellinduction factors. Thus, the ability of the methods of the presentinvention to generate purified or treated ssRNAs that result inefficient induction of iPS cells from human or animal somatic cellsfurther demonstrates the significance and breadth of this method formaking purified or treated ssRNAs for translation in living cells.

The ability of the methods of the present invention to generate treatedssRNAs that do not activate RNA sensors or RNA signaling pathways, suchas TLR3 pathways, and do not induce apoptosis pathways, even afterintroducing the treated ssRNAs into the cells 18 or more times over atleast 18 days, further demonstrates the power of the methods of thepresent invention, and the comparative advantage of these methods overother methods known in the art.

In certain embodiments, methods for treating in vitro-synthesized ssRNAswith RNase III can be performed in less than an hour, with only a fewminutes of hands-on time, and many different ssRNAs can be treatedsimultaneously, making the method easily adaptable to high-throughputproduction of purified ssRNAs. Since, in certain embodiments, certainmethods described herein primarily comprise an enzymatic step, whichmay, for example, be performed by simple pipetting steps, in someembodiments, the present method is performed unattended using alaboratory robot. Thus, the invention provides, in certain embodiments,an automated method for making purified ssRNAs for reprogramming humanor animal somatic cells to iPS cells or for reprogramming one type ofsomatic cell to another type of somatic cell.

In addition to the above, the present Applicants have also found thatpurified ssRNAs comprising modified nucleosides that are purified by anHPLC purification method can also be used for reprogramming humansomatic cells to iPS cells, as disclosed herein. However, the presentmethods using RNase III treatment are much easier, faster and moreeconomical in terms of time, materials and reagents than HPLCpurification methods for generating purified ssRNAs for reprogrammingeukaryotic somatic cells to iPS cells or for other applications.

In some preferred embodiments of the methods, compositions or kits ofthe invention, the treated RNA composition comprising ssRNA or mRNA isrepeatedly or continuously contacted with or repeatedly or continuouslyintroduced into a human or animal (e.g., mammalian) cell that is ex vivoin culture or in vivo in an organism, wherein the RNA composition iscapable of inducing a biological or biochemical effect (e.g.,reprogramming of the cell from a first differentiated state or phenotypeto a second differentiated state or phenotype), Thus, one embodiment ofthe invention is a method for inducing a biological or biochemicaleffect in a human or animal cell (e.g., mammalian cell), comprising:repeatedly or continuously introducing an RNA composition comprising oneor more ssRNAs or mRNAs encoding one or more proteins (e.g., one or moreprotein reprogramming factors, e.g., one or more transcription factors)into a human or animal cell in culture, and culturing under conditionswherein the biological or biochemical effect is induced.

In some embodiments, the biological effect comprises reprogramming acell that exhibits a first differentiated state or phenotype to a cellthat exhibits a second differentiated state of phenotype. In someembodiments, the human or mammalian cell that exhibits a firstdifferentiated state or phenotype is a somatic cell (e.g., a fibroblast,keratinocyte, or blood cell), the ssRNAs or mRNAs encode one or morereprogramming factors or iPSC induction factors selected from the groupconsisting of OCT4, SOX2, KLF4, LIN28, NANOG, and a MYC family proteinchosen from among wild-type c-MYC, mutant c-MYC(T58A), and L-MYC, andthe cell that exhibits the second differentiated state or phenotype isan iPS cell. In some embodiments, wherein the human or mammalian cellthat exhibits a first differentiated state or phenotype is a somaticcell (e.g., a fibroblast cell), said culturing comprises culturing thecells in the absence of feeder cells in the presence of at least onesmall molecule inhibitor of transforming growth factor-beta (TGF-beta orTGFβ), at least one small molecule inhibitor of mitogen-activatedprotein kinase (MAPK/ERK kinase or MEK), or at least one small moleculeinhibitor for both TGF-beta and MEK; in some of these embodiments, thecells are cultured: (i) on feeder cells; (ii) on a biological substratethat does not comprise live feeder cells (e.g., an extracellular matrixextract, e.g., a gelatinous protein mixture secreted byEngelbreth-Holm-Swarm (EHS) mouse sarcoma cells, e.g., as marketed undertradenames such as MATRIGEL™ or CULTREX BME (BD Biosciences); or one ormore biomolecules, e.g., purified human vitronectin protein); (iii)directly on a culture dish surface to which the first type of cellsadhere and grow to form a monolayer in the absence of feeder cells or abiological substrate.

One other embodiment of the present invention is a Feeder-freeReprogramming Medium consisting of Dulbecco's modified Eagle medium withnutrient mixture F-12 (DMEM/F12; Invitrogen) supplemented with 20%KNOCKOUT™ serum replacement (Invitrogen), 2 mM GLUTAMAX™-I (Invitrogen),0.1 mM non-essential amino acids solution (Invitrogen), and0.5-15—micromolar MEK signaling pathway inhibitor (e.g., STEMOLECULE™PD0325901, Stemgent, Cambridge, Mass., USA). In some embodiments, theFeeder-free Reprogramming Medium further comprises transforming growthfactor β (TGFβ) inhibitor (e.g., STEMOLECULE™ SB431542, Stemgent™). Insome embodiments, the Feeder-free Reprogramming Medium further comprisesabout 100 ng/ml basic human recombinant fibroblast growth factor. Insome embodiments, the Feeder-free Reprogramming Medium further comprisespenicillin and streptomycin antibiotics.

As shown in EXAMPLE 23, when unmodified GAUC Luc2 dsRNA or modifiedGAψC-dsRNA was added daily for two days with the respective GAUC mRNA orGAψC mRNA encoding MYOD mRNA, reprogramming of mouse mesenchymal stemcells to myoblast cells was induced only if the amount of added Luc2dsRNA was less than about 0.01% of the total mass of RNA used forreprogramming, However, when modified GAψm⁵C Luc2 dsRNA was added dailyfor two days with GAψm⁵C mRNA encoding MYOD mRNA, myoblast cells wereinduced when the Luc2 dsRNA was less than about 0.1% of the total massof RNA in the RNA composition.

Thus, one embodiment of the invention is a method for reprogramming ahuman or mammalian non-myoblast cell (e.g., a mouse mesenchymal stemcell) to a myoblast cell comprising: daily, for at least two days,introducing into non-myoblast cells an RNA composition comprising invitro-synthesized GAUC mRNA or GAψC mRNA encoding MYOD protein or afunctional fragment or variant thereof, wherein said RNA composition isat least practically free of dsRNA, and culturing under conditionswherein at least a portion of said non-myoblast cells are reprogrammedor differentiated into myoblast cells.

Thus, one other embodiment of the invention is a method forreprogramming a human or mammalian non-myoblast cell (e.g., a mousemesenchymal stem cell) to a myoblast cell comprising: daily, for atleast two days, introducing into non-myoblast cells an RNA compositioncomprising in vitro-synthesized GAψm⁵C mRNA encoding MYOD protein or afunctional fragment or variant thereof, wherein said RNA composition isat least virtually free, essentially free, or more preferablypractically free of dsRNA, and culturing under conditions wherein atleast a portion of said non-myoblast cells are reprogrammed ordifferentiated into myoblast cells.

When unmodified GAUC Luc2 dsRNA was added daily with the GAψC-mRNAsencoding ASCL1, MYT1L, NEUROD1, and POU3F2 (AMNP) reprogramming factors,neurons were induced only if the amount of added unmodified GAUC Luc2dsRNA was less than about 0.01% of the total mass of RNA used forreprogramming, and significant numbers of neurons were generated only ifthe amount of added unmodified GAUC Luc2 dsRNA was less than about0.001% of the total mass of RNA used for reprogramming. When modifiedGAψC Luc2 dsRNA was added daily with the GAψC-mRNAs encoding AMNPreprogramming factors, neurons were induced only ifpseudouridine-modified GAψC Luc2 dsRNA was less than about 0.02% of thetotal mass of RNA used for reprogramming, and significant numbers ofneurons were generated only if the amount of added unmodified GAUC Luc2dsRNA was less than about 0.004% of the total mass of RNA used forreprogramming. These results show, for certain embodiments, that thedsRNA should generally be reduced to below those levels (e.g., using theRNase III treatment methods described herein) in order to reprogramhuman fibroblasts to neuron cells as shown in EXAMPLE 24.

Thus, one other embodiment of the invention is a method forreprogramming non-neuron somatic cells (e.g., human fibroblast cells) toneuron cells, the method comprising: daily, for multiple days (e.g., forabout six or more days), introducing into non-neuron somatic cells exvivo in culture, an RNA composition comprising in vitro-synthesizedssRNA or mRNA encoding at least one protein selected from the groupconsisting of: ASCL1, MYT1L, NEUROD1 and POU3F2 or functional fragmentor variant of any thereof, wherein said RNA composition is at leastpractically free, or more preferably, extremely free or absolutely freeof dsRNA, and culturing under conditions wherein at least a portion ofsaid non-neuron somatic cells are reprogrammed or transdifferentiatedinto neuron cells.

Another embodiment of the invention is a method for reprogramming ahuman or mammalian non-cardiac fibroblast cells to a cardiac fibroblastcells, the method comprising: daily, for multiple days, introducing intohuman or mammalian fibroblasts ex vivo in culture an RNA compositioncomprising in vitro-synthesized ssRNA or mRNA encoding at least oneprotein transcription factor or reprogramming factor selected from thegroup consisting of: ETS2, MESP1, GATA4, HAND2, TBX5 and MEF2C, or afunctional fragment or variant of any thereof, wherein the RNAcomposition is practically free, extremely free or absolutely free ofdsRNA, and culturing under conditions wherein the non-cardiac fibroblastcells are reprogrammed into cardiac fibroblast cells.

One embodiment of the invention is a method for reprogramming a human ormammalian fibroblast cells to dopaminergic neuron cells, the methodcomprising: daily, for multiple days, introducing into human ormammalian fibroblasts ex vivo in culture an RNA composition comprisingin vitro-synthesized ssRNA or mRNA encoding at least one proteintranscription factor or reprogramming factor selected from the groupconsisting of: ASCL1, EN1, FOXA2, LMX1A, NURR1 and PITX3, or afunctional fragment or variant of any thereof; wherein the RNAcomposition is extremely free or absolutely free of dsRNA, and culturingunder conditions wherein the fibroblast cells are reprogrammed intodopaminergic neuron cells.

One embodiment of the invention is a method for reprogramming a human ormammalian fibroblast cells to hepatocytes, the method comprising: daily,for multiple days, introducing into human or mammalian fibroblasts exvivo in culture an RNA composition comprising in vitro-synthesized ssRNAor mRNA encoding at least one protein transcription factor orreprogramming factor selected from the group consisting of: HNF1α orfunctional fragment or variant thereof, HNF4α, FOXA1, FOXA2, FOXA3 andGATA4, or functional fragment or variant of any thereof; wherein the RNAcomposition is absolutely free of dsRNA, and culturing under conditionswherein the fibroblast cells are reprogrammed into hepatocytes.

In some preferred embodiments, the RNA composition or the invitro-synthesized ssRNA or mRNA composing the RNA composition that ispractically free of dsRNA, extremely free of dsRNA, or absolutely freeof dsRNA is less immunogenic (or induces a detectably lower immuneresponse or a detectably lower innate immune response) in said cell orin a human or animal (e.g., mammalian) tissue, organ or organismcontaining said cell than an RNA composition or the in vitro-synthesizedssRNA or mRNA composing the RNA composition that is not practically freeof dsRNA, extremely free of dsRNA, or absolutely free of dsRNA. In someembodiments, the RNA composition or the in vitro-synthesized ssRNA ormRNA composing the RNA composition is analyzed to induce a detectablylower innate immune response as detected by a method selected from thegroup consisting of: (i) detecting that repeatedly contacting themammalian cell with an amount of the modified RNA that results indetectable expression of the encoded protein after a single contactingdoes not detectably reduce expression of the protein, whereas repeatedlycontacting the mammalian cell with the same quantity of the unmodifiedRNA does detectably reduce expression of the encoded protein; (ii)detecting that the modified RNA results in a lower level ofself-phosphorylation of RNA-activated protein kinase (PKR) and/orphosphorylation of the eukaryotic translation initiation factor (eIF2α)compared to the same quantity of the unmodified RNA counterpart based onin vitro phosphorylation assays; (iii) detecting that the quantity ofone or more cytokines induced by the mammalian cell in response tounmodified RNA is higher than the quantity of said one or more cytokinesinduced by the mammalian cell in response to said modified RNAcounterpart; (iv) detecting a difference in the level of expression ofone or more dendritic cell (DC) activation markers in response to theunmodified RNA compared to the level of expression of said one or moreDC activation markers in response to the same quantity of said modifiedRNA; (v) detecting a higher relative ability of said modified RNA to actas an adjuvant for an adaptive immune response compared to the samequantity of unmodified RNA counterpart; (vi) detecting a higher level ofactivation of toll-like receptor (TLR) signaling molecules in responseto unmodified RNA compared to the same quantity of said modified RNA;and/or (vii) determining the quantity of the modified RNA to elicit animmune response measured in any of cells (i)-(vi) compared to thequantity of unmodified RNA to elicit the same immune response;particularly wherein: said one or more cytokines in (iii) are selectedfrom the group consisting of: IL-12, IFN-alpha, TNF-alpha, RANTES,MIP-1alpha, MIP-1beta, IL-6, IFN-beta, and IL-8; said DC activationmarkers in (iv) are selected from the group consisting of: CD83, HLA-DR,CD80, and CD86; and/or said TLR signaling molecules in (vi) are selectedfrom the group consisting of: TLR3, TLR7, and TLR8 signaling molecules.In some preferred embodiments the detectably lower innate immuneresponse induced by said RNA composition or the in vitro-synthesizedssRNA or mRNA composing the RNA composition that is practically free ofdsRNA, extremely free of dsRNA, or absolutely free of dsRNA compared tosaid RNA composition or the in vitro-synthesized ssRNA or mRNA composingthe RNA composition that is not practically free of dsRNA, extremelyfree of dsRNA, or absolutely free of dsRNA is at least 2-fold lowerusing at least one of said cells for determining or measuring saiddetectable decrease in immunogenicity. For example, in some embodimentsthe RNA composition or the in vitro-synthesized ssRNA or mRNA composingthe RNA composition that is practically, extremely or absolutely free ofdsRNA is analyzed to induce a detectably lower innate immune response asdescribed in U.S. Patent Application No. 20110143397, incorporatedherein by reference, particularly as described in paragraph [0262] andin “Materials and Methods for Examples 35-38” and/or as described andshown for FIGS. 22-24 therein.

One embodiment of the invention is a method for making a biologicalcomposition (e.g., an RNA composition) that is at least practically freeof dsRNA, the method comprising: treating the biological composition(e.g., an RNA composition or ssRNA or mRNA composing an RNA composition)with a dsRNA-specific protein in a buffered solution under conditionswherein the dsRNA-specific protein binds and/or reacts with dsRNAcontaminants, and then removing the dsRNA-specific protein and the boundor reacted dsRNA contaminants to generate a treated RNA preparation (ortreated ssRNA or mRNA composing the RNA composition) that is at leastpractically free of dsRNA. With respect to the methods, compositions orkits of the present invention, a “dsRNA-specific protein” herein means aprotein that is not an antibody, which protein binds and/or reacts withdsRNA with much higher affinity and specificity than it binds and/orreacts with other non-dsRNA biomolecules. In some specific embodiments,the dsRNA-specific protein is a dsRNA-specific ribonuclease (RNase). Insome preferred embodiments, the dsRNA-specific RNase is anendoribonuclease (endoRNase). Most preferably, the endoRNase of themethods, compositions or kits of the invention is RNase III.

One preferred embodiment of the invention, wherein the dsRNA-specificprotein is RNase III, is a method for making a biological composition(e.g., an RNA composition) that is substantially free, virtually free,essentially free, practically free, extremely free, or absolutely freeof dsRNA, the method comprising: contacting a biological composition(e.g., an RNA composition or ssRNA or mRNA composing an RNA composition)with RNase III in a buffered solution containing a magnesium saltcomprising magnesium cations at a concentration of about 1 mM to about 4mM under conditions wherein the RNase III binds and/or reacts with dsRNAthat is present in the solution to generate a treated biologicalcomposition that is substantially free, virtually free, essentiallyfree, practically free, extremely free, or absolutely free of dsRNA.When used to make an RNA composition that is substantially free,virtually free, essentially free, practically free, extremely free, orabsolutely free of dsRNA, this method is sometimes referred to as an“RNase III treatment” or “RNase III treatment method” herein. In somepreferred embodiments of the RNase III treatment method or embodimentsof compositions or kits comprising or for practicing the RNase IIItreatment method, the buffered solution comprises a Tris buffer (e.g.,33 mM Tris-acetate, pH 8) as the buffer, In some other embodiments, adifferent buffer that maintain the pH at about pH 7.5-8 is used. In someembodiments, a different buffer or a different pH somewhat outside ofthe range of pH 7.5-8 is used. In preferred embodiments the solutionfurther comprises a monovalent salt at a concentration of at least about50 mM, and more preferably, the solution further comprises a monovalentsalt at a concentration of about 50 mM to about 150 mM, and mostpreferably, the solution further comprises a monovalent salt at aconcentration of about 150 mM or greater than 150 mM. In someembodiments of the method, the method further comprises cleaning up thebiological composition from the RNase III and other components in thesolution. Some embodiments of the invention comprise a biologicalcomposition (e.g, an RNA composition) or a kit comprising a biologicalcomposition (e.g, an RNA composition) that is generated using the RNaseIII treatment methods described herein, wherein the biologicalcomposition (e.g, an RNA composition or ssRNA or mRNA composing an RNAcomposition) is substantially free, virtually free, essentially free,practically free, extremely free, or absolutely free of dsRNA.

Some preferred embodiments of the invention wherein the biologicalcomposition is an RNA composition comprising ssRNA or mRNA are: (i) themethod for making a biological composition that is substantially free,virtually free, essentially free, practically free, extremely free, orabsolutely free of dsRNA, (ii) a biological composition that issubstantially free, virtually free, essentially free, practically free,extremely free, or absolutely free of dsRNA made using the method, (iii)a kit comprising a biological composition that is substantially free,virtually free, essentially free, practically free, extremely free, orabsolutely free of dsRNA, or (iv) a kit for making a biologicalcomposition that is substantially free, virtually free, essentiallyfree, practically free, extremely free, or absolutely free of dsRNA,wherein said RNA composition is substantially free of dsRNA, virtuallyfree of dsRNA, essentially free of dsRNA, practically free of dsRNA,extremely free of dsRNA, or absolutely free of dsRNA, meaning,respectively, that less than about: 0.5%, 0.1%, 0.05%, 0.01%, 0.001%, or0.0002% of the RNA in the RNA composition comprises dsRNA of a sizegreater than about 40 basepairs (or greater than about 30 basepairs). Insome preferred embodiments, the biological composition comprises orconsists of an RNA composition comprising one or more invitro-synthesized ssRNAs or mRNAs (or the one or more invitro-synthesized ssRNAs or mRNAs) and the method comprises: contactingthe RNA composition or the one or more ssRNAs or mRNAs with RNase III ina buffered solution comprising divalent magnesium cations at aconcentration of about 1 mM to about 4 mM and a monovalent salt at aconcentration of at least 50 mM and incubating under conditions whereinthe RNase III binds to the dsRNA and is enzymatically active, and thencleaning up the RNA composition or the ssRNA or mRNAs from the RNase IIIand the other components, including the RNase III digestion products, togenerate a treated RNA composition or treated ssRNAs or_mRNAs that is(are) substantially free, virtually free, essentially free, practicallyfree, extremely free or absolutely free of dsRNA. In preferredembodiments of this method, treated RNA composition or treated ssRNAs ormRNAs is (are) practically free, extremely free or absolutely free ofdsRNA. In some preferred embodiments of this method, the monovalent salthas a concentration of about 50 mM to about 100 mM, about 100 mM toabout 200 mM, or about 200 mM to about 300 mM. In some preferredembodiments of the method, said cleaning up the ssRNAs or mRNAscomprises at least one step selected from: extracting with organicsolvent (e.g., phenol and/or chloroform), precipitating the ssRNAs ormRNAs with ammonium acetate, and washing the precipitate with alcohol(e.g., 70% ethanol). In preferred embodiments the cleanup does notcomprise a chromatographic column or electrophoretic gel device. In someembodiments, said cleanup comprises a gel (e.g., crosslinked dextran)filtration spin column. In certain preferred embodiments of this method,the buffered solution comprises divalent magnesium cations at aconcentration of about 1.0 mM to about 3.0 mM, or more preferably, about1.0 mM to about 2.0 mM.

In some embodiments of the method for making an RNA compositioncomprising ssRNA or mRNA that is substantially free, virtually free,essentially free, practically free, extremely free, or absolutely freeof dsRNA, method further comprises at least one step selected from amongammonium acetate precipitation, alcohol precipitation, and organicextraction (e.g., phenol and/or chloroform extraction), (e.g., each asdescribed in one or more of the Examples presented herein). In somepreferred embodiments, the RNA composition comprises ssRNA or mRNAencoding one or more proteins, In some preferred embodiments of themethod for making an RNA composition comprising ssRNA or mRNA that issubstantially free, virtually free, essentially free, practically free,extremely free, or absolutely free of dsRNA, the method does notcomprise any column chromatography (whether gravity flow or underpressure, e.g., HPLC or FPLC), electrophoresis, or other separation stepcomprising use of a resin, gel or membrane. Thus, some advantages of thepresent method for making an RNA composition comprising ssRNA or mRNAthat is substantially free, virtually free, essentially free,practically free, extremely free, or absolutely free of dsRNA are thatno such separation, chromatography, electrophoresis or specialinstrumentation is required, all of which may require special training,materials (e.g., columns, membranes), additional work and time (e.g.,packing of columns, washing of columns, special analytic methods), andcosts therefor, and which may be time consuming and require specialanalytic methods. Thus, the present method for making RNA compositionsis much easier, faster, and economical than other methods, whilegenerating RNA compositions that are equal or better for use in methodscomprising contacting the RNA compositions with a human or animal cell(e.g., to induce a biological or biochemical effect, e.g., to reprograma cell from a first differentiated state or phenotype to a seconddifferentiated state or phenotype). In view of these advantages andbenefits over methods for purification comprising a separation device(e.g., HPLC or preparative electrophoresis, we believe the presentlydescribed method for making a treated RNA composition will significantlyaccelerate work on methods for using RNA compositions comprising ssRNAor mRNA encoding one or more protein, which RNA compositions arepractically free, extremely free or absolutely free of dsRNA, to inducea biological or biochemical effect by repeatedly or continuouslyintroducing said RNA composition in to a human or animal (e.g.,mammalian) cell (e.g., a cell that is ex vivo in culture or in vivo in atissue, organ or organism)

In other embodiments of the compositions, reaction mixtures, kits andmethods of the invention, the in vitro-synthesized ssRNA does not encodea protein or polypeptide, but instead comprises at least one longnon-coding RNA (ncRNA). Thus, in some embodiments, the ssRNA exhibits asequence of at least one long ncRNA. In some embodiments of thecompositions, reaction mixtures, kits and methods of the invention, thein vitro-synthesized ssRNA exhibits a sequence of at least one longncRNA that is capable of effecting a biological or biochemical effectupon its repeated or continuous introduction into a human or animal cell(e.g., a mammalian cell). In some embodiment of compositions, kits andmethods of the invention, the ssRNA is at least one long ncRNA referredto “HOX antisense intergenic RNA” (Woo C J and Kingston R E, 2007), alsoknown as “HOTAIR,” “HOXAS,” “HOXC-AS4,” “HOXC11-AS1” or “NCRNA00072.”

Some embodiments of the invention comprise (i) a method for making abiological composition (e.g., an RNA composition) that is substantiallyfree, virtually free, essentially free, practically free, extremelyfree, or absolutely free of dsRNA, or (ii) a reaction mixture orbiological composition (e.g., a reaction mixture) that is generatedusing the method for making a biological composition that issubstantially free, virtually free, essentially free, practically free,extremely free, or absolutely free of dsRNA, or (iii) a kit comprising abiological composition that is substantially free, virtually free,essentially free, practically free, extremely free, or absolutely freeof dsRNA, or (iv) a kit for making a biological composition that issubstantially free, virtually free, essentially free, practically free,extremely free, or absolutely free of dsRNA, wherein the biologicalcomposition does not comprise an RNA composition or ssRNA or mRNAcomposing an RNA composition. With respect to these embodiments of theinvention, by “substantially free, virtually free, essentially free,practically free, extremely free, or absolutely free of dsRNA,” we meanthat the biological composition in the final solution in which it iscontacted with human or animal cells contains: less than about 5nanograms of dsRNA per ml of solution, less than about 1 nanogram ofdsRNA per ml of solution, less than about 500 picograms of dsRNA per mlof solution, less than about 100 picograms of dsRNA per ml of solution,less than about 10 picograms of dsRNA per ml of solution, or less thanabout 2 picograms of dsRNA per ml of solution, respectively. Inparticular embodiments of the method, biological composition or kitcomprising a biological composition that is substantially free,virtually free, essentially free, practically free, extremely free, orabsolutely free of dsRNA, the biological composition comprises one ormore biologicals selected from the group consisting of: double-strandedDNA (dsDNA), single-stranded DNA (ssDNA), proteins, carbohydrates,lipids, glycoproteins, lipoproteins, growth factors, cytokines, cellularextracts, extracellular matrixes, serum, biological fluids, biologicalmembranes, and media.

In some embodiments of the method for making a biological composition(e.g., an RNA composition) that is substantially free, virtually free,essentially free, practically free, extremely free, or absolutely freeof dsRNA, the method further comprises contacting the solution with oneor more deoxyribonucleases (DNases) to generate a biological compositionthat is virtually free, practically free, or extremely free of DNA,meaning that the biological composition in the final solution in whichit is contacted with human or animal cells contains less than about onenanogram of DNA per ml of solution, less than about 100 picograms of DNAper ml of solution, or less than about 10 picograms of DNA per ml ofsolution. In some embodiments, the DNAse is a “type I DNase,” meaning anendodeoxyribonuclease that digests single-stranded and double-strandedDNA to short oligonucleotides having a 5′-phosphate and a 3′-hydroxylgroup (e.g., human, bovine or porcine pancreatic DNase I). In someembodiments, the DNAse is a single-strand-specific 3′-to-5′exodeoxyribonuclease that lacks ribonuclease activity, but that digestsoligodeoxyribonucleotides having a free 3′-hydroxyl group to5′-monodeoxyribonucleotides (e.g., Escherichia coli exonuclease I). Insome embodiments, multiple DNases are used. Thus, in some embodiments ofthe biological compositions or kits comprising the a biologicalcomposition that is substantially free of dsRNA, virtually free,essentially free, practically free of dsRNA, extremely free of dsRNA, orabsolutely free of dsRNA, the biological composition or kit is alsovirtually free, practically free, or extremely free of DNA.

In certain embodiments of the methods, compositions or kits of theinvention, the RNA composition is treated or purified to be at leastvirtually dsRNA-free (e.g., virtually free of dsRNA, practically free ofdsRNA, extremely free of dsRNA, or absolutely free of dsRNA) byseparation of the ssRNA or mRNA from RNA contaminants comprising saidRNA composition using one or more chromatographic or electrophoreticseparation media (e.g., using a chromatographic or electrophoreticseparation method discussed elsewhere herein). In some preferredembodiments of the methods, compositions or kits, the RNA composition ispurified to be at least virtually dsRNA-free (e.g., virtually free ofdsRNA, practically free of dsRNA, extremely free of dsRNA, or absolutelyfree of dsRNA) by separation of the ssRNA or mRNA from RNA contaminantsby HPLC. In some preferred embodiments of the methods, compositions orkits, the in vitro-synthesized ssRNA or mRNA composing the RNAcomposition was purified (e.g., to be virtually free of dsRNA,practically free of dsRNA, extremely free of dsRNA, or absolutely freeof dsRNA) by HPLC and analyzed for purity and immunogenicity asdescribed in U.S. Patent Application No. 20110143397, incorporatedherein by reference, particularly as described in paragraph [0262] andin “Materials and Methods for Examples 35-38” and/or as described andshown for FIGS. 22-24 therein.

Still another embodiment of the invention is a method for inducing abiological or biochemical effect in a human or other mammalian cell,either ex vivo in culture or in vivo in a human or mammalian organism,comprising: repeatedly or continuously contacting the cell with the atleast practically dsRNA-free RNA composition over multiple days underconditions wherein the RNA composition is introduced into the cell and abiological or biochemical effect is induced. In some embodiments, the atleast practically dsRNA-free RNA composition comprises ssRNA or mRNAthat encodes one or more reprogramming factors and the biological orbiochemical effect comprises reprogramming the cells from a firstdifferentiated state or phenotype to a second differentiated state orphenotype. Thus, in some embodiments, the invention provides a rapid,efficient method for changing the state of differentiation or phenotypeof a human or mammalian cell. For example, in some embodiments, thepresent invention provides at least practically dsRNA-free RNAcompositions comprising ssRNA or mRNA and methods for their use toreprogram human or mammalian somatic cells to pluripotent stem cells. Insome preferred embodiments, the at least practically dsRNA-freecompositions used for said method is practically free of dsRNA,extremely free of dsRNA, or absolutely free of dsRNA.

Certain embodiments of the present invention provide ex vivo methods,and compositions and kits for rapidly and efficiently reprogramminghuman or animal cells in culture from a first differentiated state orphenotype to a second differentiated state or phenotype by repeatedly orcontinuously introducing purified or treated in vitro-synthesized mRNAsencoding multiple proteins (e.g., reprogramming factors) into the cellsfor multiple days, whereby the second differentiated state or phenotypeis induced. For example, in some embodiments, human somatic cells, suchas fibroblasts or keratinocytes, were reprogrammed (dedifferentiated) toinduced pluripotent stem cells by repeatedly introducing invitro-synthesized mRNAs encoding multiple iPSC reprogramming factorproteins into the cells daily for multiple days. In other embodiments,human non-neural somatic cells, such as fibroblasts, were reprogrammed(transdifferentiated) to neural cells by repeatedly introducing mRNAsencoding multiple neural cell reprogramming factor proteins daily formultiple days. In still other embodiments, mouse mesenchymal stem cellswere reprogrammed (differentiated) to myoblast cells by introducing mRNAencoding MYOD protein daily for two days. Thus, in some embodiments, theinvention provides general methods for reprogramming cells from a firstdifferentiated state to a second differentiated state by repeatedly orcontinuously introducing mRNA encoding one or more proteins into thecells daily for 2 or more days.

In certain embodiments, the present invention provides methods forinducing a biological or biochemical effect in a human or animal cell(e.g., a mammalian cell; e.g., a cell in culture or in vivo or in atissue, organ or organism that contains them) comprising: repeatedly orcontinuously introducing said treated and/or purified invitro-synthesized mRNAs encoding one or more proteins that is/arecapable of inducing the desired biological or biochemical effect intosaid cells. In some embodiments of the methods, the biological orbiochemical effect comprises reprogramming of a cell from a first stateof differentiation or phenotype to a second state of differentiation orphenotype. In some embodiments of the methods, the cell is a human oranimal (e.g., mammalian) immune system cell, the in vitro-synthesizedssRNA or mRNA encodes one or more proteins comprising the immunoglobulinsuperfamily, and the biological or biochemical effect comprises bindingof the one or more immunoglobulin superfamily proteins expressed on thesurface of the immune system cells to one or more exogenous proteins orpolypeptides, which exogenous proteins or polypeptide are either free orin or on the surface of a non-immune system cell, thereby initiating animmune response mechanism in response to said exogenous protein orpolypeptide. In some embodiments of the methods, the cell is an antigenpresenting cell (APC), such as a human or mammalian dendritic cell, andthe biological or biochemical effect comprises presentation of a peptidederived from said one or more proteins encoded by the invitro-synthesized ssRNA or mRNA on the surface of the APC; in certainpreferred embodiments, the composition comprising in vitro-synthesizedssRNA or mRNA does not result in production of interferon. In otherembodiments of the methods, the cell is a human or mammalian cell thatcontains a mutant gene encoding a defective protein and the biologicalor biochemical effect comprises expressing one or more proteins encodedby the in vitro-synthesized ssRNA or mRNA in said cells, therebysubstituting or compensating for the defective protein.

In some embodiments of the methods, compositions and/or kits of thepresent invention, the ssRNA or mRNA encodes a protein. In someembodiments of the methods, compositions and/or kits of the presentinvention, the ssRNA or mRNA encodes a functional protein, wherein theterm “functional” means that the protein is capable of causing abiochemical change or a biological effect (e.g., therapeutic treatment,such as a reduction of symptoms in a subject), whether direct orindirect (e.g., via a signaling pathway), in a cell in which the proteinis present or in another cell that is affected by the protein or by thecell in which the protein is expressed. In some embodiments of themethods, compositions and/or kits of the present invention, the ssRNA ormRNA encodes a transcription factor. In some embodiments of the methods,compositions and/or kits of the present invention, the ssRNA or mRNAencodes an enzyme. In some embodiments of the methods, compositionsand/or kits of the present invention, the ssRNA or mRNA encodes acluster of differentiation or CD molecule. In some embodiments of themethods, compositions and/or kits of the present invention, the ssRNA ormRNA encodes an antibody. In some embodiments of the methods,compositions and/or kits of the present invention, the ssRNA or mRNAencodes a protein that is present on or in a cell membrane. In someembodiments of the methods, compositions and/or kits of the presentinvention, the ssRNA or mRNA encodes a protein that comprises a receptorfor a signaling pathway. In some embodiments of the methods,compositions and/or kits of the present invention, the ssRNA or mRNAencodes an immune effector protein. In some embodiments of the methods,compositions and/or kits of the present invention, the ssRNA or mRNAencodes a complement protein of a vertebrate immune system. In someembodiments of the methods, compositions and/or kits of the presentinvention, the ssRNA or mRNA comprises a multiplicity of different mRNAmolecules which encode a multiplicity of different proteins.

In some embodiments, the present invention relates to compositions, kitsand rapid, efficient methods for changing the state of differentiationof a human or animal eukaryotic cell. For example, the present inventionprovides ssRNA or mRNA molecules and methods for their use to reprogramcells, such as to reprogram human or animal somatic cells to pluripotentstem cells.

In some embodiments, the present invention provides methods for changingor reprogramming the state of differentiation or differentiated state orphenotype of a human or animal cell comprising: introducing mRNAencoding at least one reprogramming factor into a cell that exhibits afirst differentiated state or phenotype to generate a reprogrammed cellthat exhibits a second differentiated state or phenotype (andcompositions and kits therefor). In some embodiments, the presentinvention provides methods for changing or reprogramming the state ofdifferentiation or differentiated state or phenotype of a human oranimal cell (e.g., a mammalian cell) comprising: repeatedly oncontinuously, over a period of at least two days, introducing mRNAencoding at least one protein reprogramming factor into a cell thatexhibits a first differentiated state or phenotype to generate areprogrammed cell that exhibits a second differentiated state orphenotype (and compositions and kits therefor). In particularembodiments, the introducing comprises introducing mRNA encoding aplurality of reprogramming factors into the cell. In some embodiments,the present invention provides methods for changing the differentiatedstate or state of differentiation of a cell comprising: introducing anmRNA encoding an iPS cell induction factor into a somatic cell togenerate a reprogrammed cell (and compositions and kits therefor). Insome embodiments, the present invention provides methods for changingthe differentiated state or state of differentiation of a cellcomprising: repeatedly on continuously, over a period of at least twodays, introducing an mRNA encoding at least one protein comprising aniPS cell induction factor into a somatic cell to generate a reprogrammedcell (and compositions and kits therefor). In certain embodiments, theintroducing comprises delivering the mRNA to the somatic cell with atransfection reagent. In certain embodiments, the introducing comprisesdelivering the mRNA to the somatic cell by electrophoresis. In someembodiments, the introducing is repeated daily for at least 3 days. Insome embodiments, the introducing is repeated daily for at least 4-8days. In some embodiments, the introducing is repeated daily for atleast 8-10 days. In some preferred embodiments, the introducing isrepeated daily for at least 10 to 18 days. In some embodiments, theintroducing is repeated daily for greater than 18 days. In someembodiments, the reprogrammed cell is a dedifferentiated cell and theprocess that occurs in this method is referred to as“dedifferentiation.” One embodiment of a dedifferentiated cell is aninduced pluripotent stem cell or iPS cell (or iPSC). In some preferredembodiments of the methods, the reprogrammed cell is an iPS cell. Infurther embodiments of the methods, the reprogrammed cell is atransdifferentiated cell and the process that occurs in this method isreferred to as “transdifferentiation.” In other embodiments, the cellthat exhibits the second state of differentiation or phenotype is adifferentiated or redifferentiated somatic cell and the process thatoccurs in this method is referred to as “differentiation” or“redifferentiation.” In some embodiments wherein the introducing isrepeated daily for at least 2 days, the mRNA encodes the protein MYOD,the cell that exhibits the first state of differentiation or firstdifferentiated state is a somatic cell (e.g., a fibroblast orkeratinocyte) or a mesenchymal stem cell, and the cell that exhibits thesecond differentiated state is a myoblast cell. In these embodiments, ifthe cell that exhibits the first differentiated state is a somatic cell(e.g., a fibroblast or keratinocyte), the process istransdifferentiation, whereas if the cell that exhibits the firstdifferentiated state is a mesenchymal stem cell, and the process isdifferentiation. In some embodiments, wherein the introducing isrepeated daily for at least 4-9 days, the mRNA encodes the proteinsASCL1, MYT1L, NEUROD1 and POU3F2, the cell that exhibits the first stateof differentiation or first differentiated state is a somatic cell(e.g., a fibroblast or keratinocyte), and the cell that exhibits thesecond differentiated state is a neural cell; in this embodiment, theprocess is transdifferentiation. In some embodiments, wherein theintroducing is repeated daily for at least 4-8 days, at least 8-10 days.at least 10 to 18 days, or for greater than 18 days, the mRNA encodesthe proteins OCT4, SOX2, KLF4, and at least one MYC protein selectedfrom the group consisting of wild-type c-MYC long, mutant c-MYC(T58A),wild-type c-MYC short and L-MYC, the cell that exhibits the first stateof differentiation or first differentiated state is a somatic cell(e.g., a fibroblast or keratinocyte), and the cell that exhibits thesecond differentiated state is an iPS cell, and the process isdedifferentiation or iPS cell induction; in some of these embodiments,the mRNA further encodes one or both of the proteins LIN28 and NANOG. Insome embodiments wherein mRNAs encoding multiple different proteins areused, the introducing comprises introducing a mixture of mRNAs encodingall of the proteins, wherein each mRNA encoding a particular protein ispresent in the same molar amount as each of the other mRNAs encodingother proteins. In some other embodiments, one or more mRNAs is presentin a different molar ratio than the other mRNAs encoding other proteins.For example, in certain embodiments wherein the mRNAs encode OCT4, SOX2,KLF4, one or both of LIN28 and NANOG, and at least one MYC proteinselected from the group consisting of wild-type c-MYC long, mutantc-MYC(T58A), wild-type c-MYC short and L-MYC, the mRNA encoding OCT4 ispresent in the mRNA mixture at approximately a 3-fold molar excesscompared to the particular mRNAs introduced that encoded SOX2, KLF4,LIN28, NANOG, and the at least one MYC family protein; in some otherembodiments, in addition to the higher molar excess of mRNA encodingOCT4, the mRNA encoding KLF4 is also present in the mRNA mixture atapproximately a 1.5-fold to 3.5-fold molar excess compared to theparticular mRNAs introduced that encode SOX2, LIN28, NANOG, and the atleast one MYC family protein,

In certain preferred embodiments of the methods for changing orreprogramming the state of differentiation or phenotype of a cell, themethod is performed without the use any exogenous protein, siRNA, orsmall molecule agent that inhibits or reduces the activation, inductionor the expression of one or more proteins in an innate immune responsepathway. For example, in some embodiments of the methods for changing orreprogramming the differentiated state or phenotype of a human or animalcell, no siRNA or protein (e.g., B18R protein), antibody or smallmolecule inhibitor of an innate immune response pathway is used for saidreprogramming. In other embodiments, the methods further comprise:treating the cells that exhibit the first differentiated state orphenotype with a protein, siRNA, or small molecule agent that inhibitsor reduces the activation, induction or expression of one or more RNAsensors or proteins in an innate immune response pathway, wherein saidtreating is prior to and/or during said introducing of an mRNA encodinga reprogramming factor. In some embodiments, the agent that inhibits orreduces the activation, induction or expression of one or more RNAsensors or proteins in an innate immune response pathway is B18Rprotein. In some other embodiments, the agent is an mRNA that encodes aprotein that inhibits or reduces the activation, induction or expressionof one or more proteins comprising an RNA sensor or innate immuneresponse pathway. In some preferred embodiments, the inhibitor is anmRNA that encodes B18R protein. In some other preferred embodiments, theinhibitor is an mRNA that encodes the Vaccinia virus E3L gene protein;in preferred embodiments, the mRNA that encodes the Vaccinia virus E3Lgene protein is introduced into the cell at the same time as the mRNAencoding one or more reprogramming factors or iPS cell induction factorsare introduced.

In certain preferred embodiments of the methods for changing orreprogramming the state of differentiation or phenotype of a cell, themethod for reprogramming is performed by adding an RNase inhibitor(e.g., SCRIPTGUARD™ RNase inhibitor, CELLSCRIPT, INC., Madison, Wis.,USA) to the media or compositions comprising ssRNA or mRNA used for saidreprogramming. Also, some preferred embodiments of compositions or kitsfor said reprogramming further comprise an RNase inhibitor.

In some preferred embodiments of the compositions, kits or methods ofthe invention, the in vitro-synthesized ssRNA or mRNA comprises a 5′ capor cap (e.g., a cap comprising 7-methylguanine) on its 5′ terminus and apoly(A) tail on its 3′ terminus. In some embodiments, the 5′ cap isincorporated into the in vitro-synthesized ssRNA or mRNAco-transcriptionally by use of a dinucleotide cap analog during in vitrotranscription. In some embodiments the 5′ cap is incorporated into thein vitro-synthesized ssRNA or mRNA post-transcriptionally by incubatinguncapped ssRNA obtained from an in vitro transcription reaction with acapping enzyme comprising RNA guanyltransferase activity. In someembodiments, the 5′ cap further comprises a 5′-terminal penultimatenucleotide that exhibits a 2′-O-methyl group on its ribose moiety; insome of these embodiments, the 2′-O-methyl group is incorporated intothe in vitro-synthesized ssRNA or mRNA using RNA 2′-O-methyltransferase.In some preferred embodiments, the in vitro-synthesized ssRNA or mRNAfurther exhibits one or more sequences selected from among anuntranslated region or UTR (e.g., a UTR which further enhancestranslation of protein in a cell into which the ssRNA or mRNA isintroduced, e.g., a 5′ UTR and/or 3′ UTR of a Xenopus, human or othermammalian alpha- (α-) globin or beta-(β-) globin mRNA, or a UTR sequenceexhibited by tobacco etch virus (TEV) RNA), a KOZAK sequence, atranslation start codon, and a translation stop codon.

In particular embodiments of the methods, compositions or kits of theinvention, the ssRNA or mRNA is polyadenylated. In some embodiments, thessRNA or mRNA comprises a poly-A tail of about 50-200 nucleotides inlength. In other embodiments, the ssRNA or mRNA comprises a poly-A tail100-200 nucleotides in length. In other embodiments, the ssRNA or mRNAcomprises a poly-A tail greater than 200 nucleotides in length. In somepreferred embodiments, the ssRNA or mRNA comprises a poly-A tail ofabout 150-200 nucleotides in length. In some embodiments, the ssRNA ormRNA is made by synthesizing the poly-A tail by in vitro transcriptionof a DNA template that comprises a terminal oligo(dT) sequence that iscomplementary to the poly-A tail. In some preferred embodiments, thessRNA or mRNA is made by post-transcriptional polyadenylation of the3′-terminus of the mRNA ORF from an IVT reaction using a poly(A)polymerase (e.g., poly(A) polymerase derived from E. coli orSaccharomyces cerevisiae; or a poly(A) polymerase from a commercialsource, e.g., A-PLUS™ poly(A) polymerase, CELLSCRIPT, INC., Madison,Wis. 53713, USA). Unless otherwise specifically stated with respect to aparticular method, the invention is not limited to use of a particularpoly(A) polymerase, and any suitable poly(A) polymerase can be used. Theinvention is not limited to particular methods described herein forpolyadenylating a ssRNA for use in a method, or for making a compositionor kit of the invention. Any suitable method in the art may be used forsaid polyadenylating.

In further embodiments of the methods, compositions or kits of theinvention, the ssRNA or mRNA comprises capped mRNA. In certain preferredembodiments of the methods, compositions and kits, the ssRNA or mRNA isa population of ssRNA or mRNA molecules, the population having greaterthan 99% capped ssRNA or mRNA. In preferred embodiments of the methods,the capped mRNA exhibits a cap with a cap1 structure, wherein the2′position of the ribose of the penultimate nucleotide to the 5′ capnucleotide is methylated.

In some embodiments of the methods, compositions or kits of theinvention (e.g., for reprogramming a human or animal cell), the ssRNA ormRNA exhibits a 5′ cap comprising 7-methylguanosine or 7-methylguanine.In some embodiments of the methods, compositions or kits, the ssRNA ormRNA exhibits an anti-reverse cap analog (ARCA). In some embodiments,the mRNA exhibits a phosphorothioate cap analog, also referred to as a“thio-ARCA” herein (Grudzien-Nogalska E et al., 2007; Kowalska J et al.2008). In some embodiments, the ssRNA or mRNA further comprises a 5′ capthat has a cap1 structure, wherein the 2′ hydroxyl of the ribose of the5′ penultimate nucleotide is methylated (e.g., obtained by methylationusing a SCRIPTCAP™ 2′-O-methyltransferase kit or using a the2′-O-methylation components of the T7 mSCRIPT™ standard mRNA productionsystem (CELLSCRIPT, INC., Madison, Wis., USA). In some embodiments, thessRNA or mRNA exhibits said 5′cap are synthesized: (i)co-transcriptionally, by incorporation of an anti-reverse cap analog(ARCA) during in vitro transcription of the ssRNA molecules (e.g., usinga MESSAGEMAX™ T7 ARCA-capped message transcription kit, CELLSCRIPT,INC.); or (ii) post-transcriptionally (e.g., using T7 mSCRIPT™ standardmRNA production system, CELLSCRIPT, INC.) with a capping enzyme system,by incubating in vitro-transcribed ssRNA molecules under conditionswherein the in vitro-transcribed ssRNA molecules are 5′-capped,including wherein the capping enzyme system results in methylation ofthe 2′ hydroxyl of the ribose in the 5′ penultimate nucleotide. In somepreferred embodiments, the ssRNA molecules are capped using a cappingenzyme comprising RNA guanyltransferase and RNA 2′-O-methyltransferase.In some preferred embodiments, the ssRNA or mRNA is significantly freeof uncapped RNA molecules that exhibit a 5′-triphosphate group (whichare considered to be one type of “contaminant RNA molecules” herein). Incertain embodiments, the ssRNA or mRNA consists of a population of ssRNAor mRNA molecules, the population having: (i) greater than 80% cappedssRNA or mRNA molecules; (ii) greater than 90% capped ssRNA or mRNAmolecules; (iii) greater than 95% capped ssRNA or mRNA molecules; (iv)greater than 98% capped ssRNA or mRNA molecules; (v) greater than 99%capped ssRNA or mRNA molecules; or (vi) greater than 99.9% capped ssRNAor mRNA molecules. In some embodiments of the compositions, kits ormethods wherein the ssRNA or mRNA also comprises contaminant uncappedRNA molecules that exhibit a 5′-triphosphate group (e.g., in embodimentswherein the ssRNA or mRNA used for said introducing of ssRNA or mRNAencoding at least one reprogramming factor into a cell that exhibits afirst differentiated state or phenotype is capped co-transcriptionallyusing a cap analog), the ssRNA or mRNA used in the method for saidintroducing is first incubated with an alkaline phosphatase (e.g.,NTPhosphatase™, Epicentre Technologies, Madison, Wis., USA) or with RNA5′polyphosphatase (CELLSCRIPT, INC., Madison, Wis. or EpicentreTechnologies) to remove the 5′-triphosphate group from the contaminantuncapped RNA molecules; in some of these embodiments, the ssRNA or mRNAthat is treated with RNA 5′polyphosphatase is further treated withTERMINATOR™ 5′-phosphate-dependent exonuclease (Epicentre Technologies)or Xm1 exoribonuclease to digest contaminant uncapped RNA molecules thatexhibit a 5′-monophosphate group.

In some preferred embodiments of the methods, compositions and kits ofthe invention for reprogramming a human or animal cell, the ssRNA ormRNA exhibits at least one heterologous 5′ UTR sequence, Kozak sequence,IRES sequence, or 3′ UTR sequence that results in greater translation ofthe mRNA into at least one protein reprogramming factor in the human oranimal cells compared to the same mRNA that does not exhibit saidrespective sequence. In some particular embodiments of the methods,compositions and kits, the 5′ UTR or 3′ UTR is a sequence exhibited by aXenopus or human alpha- (α-) globin or beta- (β-) globin mRNA, orwherein the 5′ UTR is a sequence exhibited by tobacco etch virus (TEV)RNA.

In certain embodiments of the methods, compositions or kits of theinvention, except for the nucleotides comprising the cap, the ssRNA ormRNA comprises only the canonical ribonucleosides G, A, C and U. Inadditional embodiments, the ssRNA or mRNA comprises pseudouridine inplace of uridine. In some embodiments of the methods, compositions orkits for reprogramming a human or animal cell, the ssRNA or mRNAcomprises at least one modified ribonucleoside selected from the groupconsisting of pseudouridine (Ψ), 1-methyl-pseudouridine (m¹Ψ),5-methylcytidine (m⁵C), 5-methyluridine (m⁵U), 2′-O-methyluridine (Um orm^(2′-O)U), 2-thiouridine (s²U), and N⁶-methyladenosine (m⁶A) in placeof at least a portion of the corresponding unmodified canonicalribonucleoside. In some embodiments of the methods, compositions or kitsof the invention wherein the ssRNA or mRNA comprises at least onemodified ribonucleoside, the at least one modified ribonucleoside isselected from the group consisting of: (i) pseudouridine (Ψ),1-methyl-pseudouridine (m¹Ψ), 5-methyluridine (m⁵U), 2′-O-methyluridine(Um or m²′TU), and 2-thiouridine (s²U) in place of all or almost all ofthe canonical uridine residues; (ii) 5-methylcytidine (m⁵C) in place ofall or almost all of the canonical cytidine residues; and/or (iii)N⁶-methyladenosine (m⁶A) in place of all or almost all of the canonicaladenosine residues. In other embodiments, only a portion of a canonicalribonucleoside is replaced by the corresponding modified ribonucleoside,wherein a portion means 1-25%, 25-50%, or 50-99% of the canonicalribonucleoside is replaced. In some preferred embodiments of themethods, compositions or kits of the invention wherein the ssRNA or mRNAmolecules comprise at least one modified ribonucleoside, the at leastone modified ribonucleoside consists of pseudouridine (Ψ) in place ofall or almost all of the canonical uridine residues, and/or5-methylcytidine (m⁵C) in place of all or almost all of the canonicalcytidine residues. In some other embodiments, only a portion of thecanonical uridine residues are replaced by pseudouridine residues and/oronly a portion of the canonical cytidine residues are replaced by5-methylcytidine residues, wherein a portion means 1-25%, 25-50%, or50-99% of one or both canonical ribonucleosides are replaced.

In some embodiments of the methods, compositions or kits wherein thessRNA or mRNA comprises at least one modified ribonucleoside, the ssRNAor mRNA is synthesized by in vitro transcription (IVT) of a DNA templatethat encodes each said at least one protein or polypeptide reprogrammingfactor using an RNA polymerase that initiates said transcription from acognate RNA polymerase promoter that is joined to said DNA template andribonucleoside 5′ triphosphates (NTPs) comprising at least one modifiedribonucleoside 5′ triphosphate selected from the group consisting ofpseudouridine 5′ triphosphate (ΨTP), 1-methyl-pseudouridine 5′triphosphate (m¹ΨTP), 5-methylcytidine 5′ triphosphate (m⁵CTP),5-methyluridine 5′ triphosphate (m⁵UTP), 2′-O-methyluridine 5′triphosphate (UmTP or m^(2′-O)UTP), 2-thiouridine 5′ triphosphate(s²UTP), and N⁶-methyladenosine 5′ triphosphate (m⁶ATP). In somepreferred embodiments, the modified NTP is used in place of all oralmost all of the corresponding unmodified NTP in the IVT reaction(e.g., ΨTP, m¹ΨTP, m⁵UTP, m^(2′-O)UTP or s²UTP in place of UTP: m⁵CTP inplace of CTP; or m⁶ATP in place of ATP) (e.g., using a T7 mSCRIPT™standard mRNA production system (CELLSCRIPT, INC., Madison, Wis., USA),wherein the canonical NTP is replaced by the corresponding modifiedNTP).

In other preferred embodiments of the methods, compositions or kits forreprogramming a human or animal cell, the ssRNA or mRNA does not containa ribonucleoside comprising a modified nucleic acid base, other than themodified nucleic acid base (e.g., the 7-methylguanine base) comprisingthe 5′ cap nucleotide (or, e.g., if the ssRNA or mRNA was synthesizedusing a dinucleotide cap analog, possibly also including a modified basein the 5′ penultimate nucleoside). Thus, in some embodiments of themethods, compositions or kits for reprogramming a human or animal cell,except for the ribonucleoside(s) comprising the 5′ cap, the ssRNA ormRNA comprises only the canonical ribonucleosides G, A, C and U. In someembodiments of the methods, compositions or kits for reprogramming ahuman or animal cell, the ssRNA or mRNA is synthesized by in vitrotranscription (IVT) of a DNA template that encodes each said at leastone protein or polypeptide reprogramming factor using the canonicalNTPs: GTP, ATP, CTP and UTP (e.g., using a T7 mSCRIPT™ standard mRNAproduction system (CELLSCRIPT, INC., Madison, Wis., USA).

Thus, one preferred embodiment of the invention is a method forreprogramming a eukaryotic cell (e.g., a human or animal cell, e.g., amammalian cell) that exhibits a first differentiated state or phenotypeto a cell that exhibits a second differentiated state or phenotype,comprising: repeatedly or continuously introducing a compositioncomprising in vitro-synthesized ssRNA or mRNA encoding a reprogrammingfactor into a cell that exhibits a first differentiated state orphenotype to generate a reprogrammed cell that exhibits a seconddifferentiated state or phenotype, which ssRNA or mRNApredominantlyconsists of only unmodified nucleic acid bases (i.e., the canonicalnucleic acid bases: guanine, adenine, cytosine, and uracil), except forthe base comprising the 5′ cap nucleotide or, potentially, the base ofthe 5′ penultimate nucleoside which is linked to the cap nucleotide.Said another way, in these embodiments of the method, the ssRNA or mRNApredominantly consists of only the canonical nucleosides guanosine,adenosine, cytidine and uridine, except for the 5′ cap nucleotide, andthe 5′ penultimate nucleoside when the ssRNA or mRNA molecules exhibit acap1 cap structure (e.g., wherein the ssRNA, mRNA or precursor thereofwas synthesized using only or predominantly GTP, ATP, CTP and UTP duringin vitro transcription). In some embodiments, the ssRNA or mRNA issynthesized in vitro. In some embodiments of this method, the cell thatexhibits the second differentiated state or phenotype is an iPS cell. Inpreferred embodiments of the methods, compositions or kits usingunmodified ssRNA or mRNA, the ssRNA or mRNA is absolutely free of dsRNA.In additional embodiments, although the mRNA comprises almost entirelyunmodified ribonucleosides except for the 5′ cap, the ssRNA or mRNA cancomprise certain modifications for a particular purpose, including amodified internucleoside linkage, such as a phosphorothioate,phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkage(e.g., to provide resistance of the mRNA molecules to nucleases or otherenzymes that are capable of degrading canonical phosphate linkages).

By “substantially free of dsRNA” we mean that less than about 0.5% ofthe total mass or weight of the ssRNA (or the mRNA, e.g., encoding oneor more reprogramming factors or an iPS cell induction factors) iscomposed of dsRNA of a size greater than about 40 basepairs in length.By “virtually free of dsRNA” we mean that less than about 0.1% of thetotal mass or weight of the RNA comprising the ssRNA or mRNA (e.g.,encoding one or more reprogramming factors or an iPS cell inductionfactors) is composed of dsRNA of a size greater than about 40 basepairsin length. By “essentially free of dsRNA” we mean less than 0.05% of thetotal mass or weight of the ssRNA (or the mRNA, e.g., encoding one ormore reprogramming factors or an iPS cell induction factors) is composedof dsRNA of a size greater than about 40 basepairs in length. By“practically free of dsRNA” we mean that less than about 0.01% of thetotal mass or weight of the RNA comprising the ssRNA or mRNA (e.g,encoding one or more reprogramming factors or an iPS cell inductionfactors) is composed of dsRNA of a size greater than about 40 basepairsin length. By “extremely free of dsRNA” we mean that less than about0.001% of the total mass or weight of the RNA comprising the ssRNA ormRNA (e.g., encoding one or more reprogramming factors or an iPS cellinduction factors) is composed of dsRNA of a size greater than about 40basepairs in length. By “absolutely free of dsRNA” we mean that lessthan about 0.0002% of the total mass or weight of the RNA comprising thessRNA or mRNA (e.g., encoding one or more reprogramming factors or aniPS cell induction factors) is composed of dsRNA of a size greater thanabout 40 basepairs in length. In some embodiments, the amount of dsRNA(e.g., the amount of detectable dsRNA) of a size greater than about 40basepairs in length is assayed by dot blot immunoassay using adsRNA-specific antibody (e.g., the J2 dsRNA-specific antibody or the K1dsRNA-specific antibody from English & Scientific Consulting, Szirák,Hungary) using standards of known quantity of dsRNA, as describedherein, or using another assay that gives equivalent results to theassay described herein. It shall be understood herein that the resultsof the dot blot immunoassays using the J2 dsRNA-specific antibody willbe based on comparing the assay results of the ssRNA or mRNA that isintended for introducing into a human or animal cell, organism orsubject with the assay results of J2 dsRNA-specific antibody dot blotimmunoassays performed at the same time with dsRNA standards comprisingknown quantities of dsRNA of the same or equivalent size and J2 antibodybinding.

In some other embodiments, the amounts and relative amounts ofnon-contaminant mRNA molecules and RNA contaminant molecules (or aparticular RNA contaminant, e.g., a dsRNA contaminant) may be determinedby HPLC or other methods used in the art to separate and quantify RNAmolecules. In some other embodiments, the amounts and relative amountsof non-contaminant mRNA molecules and RNA contaminant molecules (or aparticular RNA contaminant, e.g., a dsRNA contaminant) is determinedusing a specific quantitative assay for a particular contaminant (e.g.,dsRNA) in a known about of total RNA. In some other embodiments, theamount of dsRNA contaminants of a size greater than about 40 basepairsin length is determined based on measuring the A₂₆₀ absorbance of allcolumn chromatography fractions or all agarose or polyacrylamide gelelectrophoresis fractions from chromatography or electrophoresis,respectively, of a sufficient quantity of in vitro-synthesized or invitro-transcribed ssRNA so that the absorbance of dsRNA contaminants inall fractions comprising RNA of a size other than the fraction orfractions confirmed to contain only RNA of the correct size and sequenceas the ssRNA or mRNA of interest so that the appropriate purity level(e.g., substantially free, virtually free, essentially free, practicallyfree, extremely free, or absolutely free will be capable of beingmeasured. In preferred embodiments of the methods, compositions or kits,the ssRNA or mRNA encoding a reprogramming factor or an iPS cellinduction factor is extremely free or absolutely free of dsRNA.

In preferred embodiments of the methods, compositions or kits, includingwherein the ssRNA or mRNA comprises a modified ribonucleoside or, exceptfor the cap, only unmodified ribonucleosides, the ssRNA or mRNA (e.g.,encoding a reprogramming factor or an iPS cell induction factor) isvirtually free, essentially free, practically free, extremely free, orabsolutely free of detectable dsRNA.

In general, the level of dsRNA contaminant in the RNA compositioncomprising mRNA encoding at least one protein that results in an innateimmune response, cellular toxicity or cell death depends upon severalfactors, such as the duration of the period of repeatedly orcontinuously contacting the cell with the RNA composition comprising themRNA required to cause the biological or biochemical effect, the amountof mRNA in said composition, and the nucleotides composing said mRNA(e.g., whether the mRNA comprises modified nucleotides, e.g., the mRNAcomprises GAψC or GAψm⁵C nucleotides, or only GAUC unmodifiednucleotides).

Thus, one preferred embodiment of the invention is a method for changingor reprogramming the state of differentiation or differentiated state orphenotype of a human or animal cell comprising: introducing ssRNA ormRNA encoding a reprogramming factor, which ssRNA or mRNA is at leastpractically free of dsRNA, into a cell that exhibits a firstdifferentiated state or phenotype to generate a reprogrammed cell thatexhibits a second differentiated state or phenotype. Another preferredembodiment is a method for changing or reprogramming the state ofdifferentiation or differentiated state or phenotype of a human oranimal cell comprising: introducing ssRNA or mRNA encoding areprogramming factor, which ssRNA or mRNA is practically free of dsRNA,into a cell that exhibits a first differentiated state or phenotype togenerate a reprogrammed cell that exhibits a second differentiated stateor phenotype. Still another preferred embodiment is a method forchanging or reprogramming the state of differentiation or differentiatedstate or phenotype of a human or animal cell comprising: introducingssRNA or mRNA encoding a reprogramming factor, which ssRNA or mRNA isextremely free of dsRNA, into a cell that exhibits a firstdifferentiated state or phenotype to generate a reprogrammed cell thatexhibits a second differentiated state or phenotype. Still anotherpreferred embodiment is a method for changing or reprogramming the stateof differentiation or differentiated state or phenotype of a human oranimal cell comprising: introducing ssRNA or mRNA encoding areprogramming factor, which ssRNA or mRNA is absolutely free of dsRNA,into a cell that exhibits a first differentiated state or phenotype togenerate a reprogrammed cell that exhibits a second differentiated stateor phenotype. In particular embodiments of the methods, the introducingcomprises introducing ssRNA or mRNA encoding a plurality ofreprogramming factors into the cell. In some embodiments, the presentinvention provides methods for changing the differentiated state orstate of differentiation of a cell comprising: introducing ssRNA or mRNAencoding at least one iPS cell induction factor, which ssRNA or mRNA isvirtually free, essentially free, practically free, extremely free orabsolutely free of dsRNA, into a somatic cell to generate a reprogrammedcell. In certain embodiments of the methods, the introducing comprisesdelivering the ssRNA or mRNA to the somatic cell with a transfectionreagent. In some embodiments, the introducing is repeated daily for atleast 3 days. In some preferred embodiments of the methods, theintroducing is repeated daily for at least 4 to 8 days, 8 to 10 days, orfor 10 to 18 days. In some embodiments, the introducing is repeateddaily for greater than 18 days. In some embodiments, the reprogrammedcell is a dedifferentiated cell and the process that occurs in thismethod is referred to as “dedifferentiation.” One embodiment of adedifferentiated cell is an induced pluripotent stem cell or iPS cell(or iPSC). In some preferred embodiments of the methods, thereprogrammed cell is an iPS cell. In further embodiments of the methods,the reprogrammed cell is a transdifferentiated cell and the process thatoccurs in this method is referred to as “transdifferentiation.” In otherembodiments, the cell that exhibits the second state of differentiationor phenotype is a differentiated or redifferentiated somatic cell andthe process that occurs in this method is referred to as“differentiation” or “redifferentiation.” In certain preferredembodiments of the methods for changing or reprogramming the state ofdifferentiation or phenotype of a cell, the method is performed withoutthe use any exogenous protein, siRNA, or small molecule agent thatinhibits or reduces the activation, induction or the expression of oneor more proteins in an innate immune response pathway. Thus, in someembodiments of the methods for changing or reprogramming thedifferentiated state or phenotype of a human or animal cell, no siRNA orprotein (e.g., B18R protein), antibody or small molecule inhibitor of aninnate immune response pathway is used for said reprogramming. In otherembodiments, the methods further comprise: treating the cells thatexhibit the first differentiated state or phenotype with a protein,siRNA, or small molecule agent that inhibits or reduces the activation,induction or expression of one or more RNA sensors or proteins in aninnate immune response pathway, wherein said treating is prior to and/orduring said introducing of an mRNA encoding a reprogramming factor. Insome embodiments, the agent that inhibits or reduces the activation,induction or expression of one or more RNA sensors or proteins in aninnate immune response pathway is B18R protein.

In some other embodiments, the agent is an Agent mRNA that encodes aprotein that inhibits or reduces the activation, induction or expressionof one or more proteins comprising an RNA sensor or innate immuneresponse pathway. In some preferred embodiments, the inhibitor is anAgent mRNA that encodes B18R protein. In some other preferredembodiments, the inhibitor is an Agent mRNA that encodes the Vacciniavirus E3L gene protein; in preferred embodiments, the Agent mRNA thatencodes the Vaccinia virus E3L gene protein is introduced into the cellat the same time as the mRNA encoding one or more reprogramming factorsor iPS cell induction factors are introduced. In preferred embodimentsof these methods, the Agent mRNA is capped. In some embodiments, greaterthan 90% of the RNA molecules comprising the Agent mRNA are capped. Inpreferred embodiments, greater than 99% of the RNA molecules comprisingthe Agent mRNA are capped. In some preferred embodiments of theseembodiments, the Agent mRNA exhibits a cap with a cap1 structure,meaning that the 2′ hydroxyls of the ribose of the 5′ penultimatenucleotide of the RNA molecules comprising the Agent mRNA aremethylated. In some embodiments of these methods, the Agent mRNA ispolyadenylated. In preferred embodiments of these methods, the AgentmRNA exhibits a poly-A tail consisting of at least 50 A residues. Insome preferred embodiments of these methods, the poly-A tail consists ofat least 100-200 A residues. In some preferred embodiments of thesemethods, the Agent mRNA exhibits at least one heterologous 5′ UTRsequence, Kozak sequence, IRES sequence, or 3′ UTR sequence that resultsin greater translation of the mRNA into at least one proteinreprogramming factor in the human or animal cells compared to the sameAgent mRNA that does not exhibit said respective sequence. In someparticular embodiments of these methods, the 5′ UTR or 3′ UTR is asequence exhibited by a Xenopus or human alpha- (α-) globin or beta-(β-) globin mRNA, or wherein the 5′ UTR is a sequence exhibited bytobacco etch virus (TEV) RNA. In some preferred embodiments of thesemethods, the Agent mRNA comprises or consists of at least one modifiednucleoside selected from the group consisting of pseudouridine (Ψ),1-methyl-pseudouridine (m¹Ψ), 5-methylcytidine (m⁵C), 5-methyluridine(m⁵U), 2′-O-methyluridine (Um or m^(2′-O)U), 2-thiouridine (s²U), andN⁶-methyladenosine (m⁶A) in place of at least a portion of thecorresponding unmodified canonical ribonucleoside. In some preferredembodiments, the at least one modified ribonucleoside is selected fromthe group consisting of: (i) pseudouridine (Ψ), 1-methyl-pseudouridine(m¹Ψ), 5-methyluridine (m⁵U), 2′-O-methyluridine (Um or m^(2′-O)U), and2-thiouridine (s²U) in place of all or almost all of the canonicaluridine residues; (ii) 5-methylcytidine (m⁵C) in place of all or almostall of the canonical cytidine residues; and/or (iii) N⁶-methyladenosine(m⁶A) in place of all or almost all of the canonical adenosine residues.In other embodiments of these methods, only a portion of a canonicalribonucleoside is replaced by the corresponding modified ribonucleoside,wherein a portion means 1-25%, 25-50%, or 50-99% of the canonicalribonucleoside is replaced. In other preferred embodiments, the at leastone modified ribonucleoside consists of pseudouridine (Ψ) in place ofall or almost all of the canonical uridine residues, and/or5-methylcytidine (m⁵C) in place of all or almost all of the canonicalcytidine residues. In some other embodiments, only a portion of thecanonical uridine residues are replaced by pseudouridine residues and/oronly a portion of the canonical cytidine residues are replaced by5-methylcytidine residues, wherein a portion means 1-25%, 25-50%, or50-99% of one or both canonical ribonucleosides are replaced. In otherpreferred embodiments of these methods, except with respect to thenucleosides comprising the 5′ cap, the mRNA consists of only unmodifiedcanonical G, A, C and U nucleosides.

In preferred embodiments of the methods, compositions, or kits of theinvention, the mRNA is extremely or absolutely free of dsRNA. Thus,since the mRNA used in the methods herein (or a precursor to the mRNA,such as in vitro-transcribed RNA (or IVT-RNA) prior to capping and/orpolyadenylation) is preferably ssRNA, we sometimes refer to the mRNAherein as an “RNA composition comprising ssRNA molecules”, an “RNAcomposition”, or “ssRNA molecules”; therefore, whenever the terms “RNAcomposition comprising ssRNA molecules”, “RNA composition” or “ssRNAmolecules” are used herein with respect to a method, composition or kitcomprising or for reprogramming a somatic cell to an iPS cell, thoseterms shall be understood to mean the “mRNA encoding a reprogrammingfactor or an iPS cell induction factor,” including wherein the mRNAencodes a plurality of reprogramming factors or an iPS cell inductionfactors. Thus, in some preferred embodiments, the RNA composition orssRNA or mRNA is absolutely free of dsRNA, meaning, for example, thatfor each one microgram or 1,000,000 picograms of RNA in the RNAcomposition, greater than 999,998 picograms comprises ssRNA and lessthan 2 picograms is dsRNA of a size greater than about 40 basepairs inlength (e.g., when assayed by immunoassay using the J2 dsRNA-specificantibody (English & Scientific Consulting, Szirák, Hungary) as describedherein or using another assay that gives equivalent results to the assaydescribed herein).

In some specific embodiments, the dsRNA-specific RNase is anexoribonuclease (exoRNase). In some specific embodiments, thedsRNA-specific RNase is an exoribonuclease (e.g., a 3′-to-5′exoribonuclease, e.g., a Lassa virus exoRNase, Qi X et al., 2010; orcoronavirus exoRNase, Hastie K M et al., 2011).

Without being bound by theory, the applicants found that, underconditions used, certain commercially antibodies (e.g., Schönborn J etal. 1991, Lukacs N 1994, Lukacs N. 1997; e.g., the J2 antibody fromEnglish & Scientific Consulting, Szirák, Hungary) that binds dsRNA,while very useful for certain dsRNA specific assays, did not appear toconsistently remove sufficient amounts of dsRNA from ssRNA or mRNA or aprecursor thereof for use in a method for making an purified or treatedRNA composition for a composition, kit or method of the presentinvention, and, therefore, such dsRNA-specific antibodies are notincluded within definition of a dsRNA-specific protein herein. However,without being bound by theory, the applicants believe that it may bepossible to generate one or more other dsRNA-specific antibodies, whichcould potentially be used, separately or in combination to make apurified or treated RNA composition.

In some embodiments, a combination of any of the above described methodsis used. Thus, any one or more particular methods for generating mRNAthat is substantially free, virtually free, essentially free,practically free, extremely free or absolutely free of dsRNA can be usedin addition to or in conjunction with any other method for generatingmRNA that is substantially free, virtually free, essentially free,practically free, extremely free or absolutely free of dsRNA. Thus, forexample, although a method comprising contacting an in vitro-synthesizedssRNA with a dsRNA-specific antibody does not appear to generate a ssRNAthat is substantially free, virtually free, essentially free,practically free, extremely free or absolutely free of dsRNA under theconditions used herein, in some embodiments, said method is used inaddition to a method comprising HPLC or the RNase III treatment methoddescribed herein to generate ssRNA (e.g., mRNA) that is substantiallyfree, virtually free, essentially free, practically free, extremely freeor absolutely free.

In some preferred embodiments wherein the dsRNA-specific protein isRNase III, the method comprises: contacting the mRNA (or precursorthereof) with the RNase III in a buffered solution comprising divalentmagnesium cations at a concentration of about 1 mM to about 4 mM and amonovalent salt at a concentration of at least 50 mM and incubatingunder conditions wherein the RNase III binds to the dsRNA and isenzymatically active, and then cleaning up the mRNA (or precursorthereof) from the RNase III and the other components, including theRNase III digestion products, to generate a treated mRNA (or precursorthereof) that is substantially free, virtually free, essentially free,practically free, extremely free or absolutely free of dsRNA. In certainpreferred embodiments of this method, the buffered solution comprisesdivalent magnesium cations at a concentration of about 1 mM to about 3mM, about 2.0 mM to about 4.0 mM, about 2 mM to about 3 mM, or about 2mM. In some preferred embodiments of this method, the monovalent salthas a concentration of about 50 mM to about 100 mM, about 100 mM toabout 200 mM, or about 200 mM to about 300 mM. Still further, the mRNA(or precursor thereof) can be extracted with phenol-chloroform,precipitated using ammonium acetate or purified by chromatography orother means as described elsewhere herein.

In some preferred embodiments (e.g., wherein the dsRNA-specific proteinis RNase III), the method comprises: contacting the ssRNA or mRNA (orprecursor thereof) with the dsRNA-specific protein (e.g., RNase III) ina buffered solution that contains a monovalent salt at a concentrationof at least 50 mM (and more preferably, about 50 to about 150 mM, orabout 150 mM to about 300 mM) but which lacks divalent magnesiumcations, and incubating under conditions wherein the dsRNA-specificprotein (e.g., RNase III) binds to the dsRNA but is not enzymaticallyactive, and then cleaning up the ssRNA or mRNA (or precursor thereof)from the dsRNA-specific protein (e.g., RNase III), at least some ofwhich is bound to the dsRNA, and from the other components, to generatessRNA or mRNA that is substantially free, virtually free, essentiallyfree, practically free, extremely free or absolutely free of dsRNA. Inthis embodiment, the present researchers utilize the very tight andspecific binding of the dsRNA-specific protein (e.g., RNase III) fordsRNA, while performing the incubation in the absence of divalentmagnesium cations so that the dsRNA-specific protein (e.g., RNase III)is not enzymatically active. Thus, in some embodiments, thedsRNA-specific protein (e.g., RNase III) is used as a binding agent forthe dsRNA, which is then removed from the mRNA (or precursor thereof) byone of several means (e.g., by using an antibody that binds to thedsRNA-specific protein (e.g., RNase III) and/or an antibody that bindsto the dsRNA (e.g., a dsRNA-specific antibody such as the J2 antibody;English and Scientific Consulting, Szirák, Hungary), which in turn canbe precipitated using commercially available particles (e.g., magneticparticles or beads) to which protein A or protein G is attached toprecipitate the antibody that is bound to the dsRNA-specific protein(e.g., RNase III), thereby purifying the mRNA (or precursor thereof). Insome embodiments wherein a dsRNA-specific protein (e.g., RNase III) isused as a binding agent for the dsRNA, the dsRNA-specific protein (e.g.,RNase III) is covalently derivatized with an affinity-binding molecule(e.g., biotin, or e.g., any other affinity-binding small molecule (e.g.,preferably a small molecule) known in the art), which covalentderivatization does not abolish dsRNA binding by the protein or changethe specificity of the dsRNA-specific protein for binding dsRNA. In someembodiments of the methods, compositions or kits, the derivatizeddsRNA-specific protein (e.g., biotin-derivatized or biotinylateddsRNA-specific protein, e.g., biotinylated RNase III) is removed bycontacting a solution containing the derivatized dsRNA-specific proteinwith a surface (e.g., magnetic particles or beads) that comprisesanother molecule that tightly and specifically binds the derivatizeddsRNA-specific protein, including the derivatized dsRNA-specific proteinthat is bound to dsRNA contaminants; for example, in one specificembodiment, a solution containing biotinylated RNase III which wascontacted with an RNA composition comprising ssRNA or mRNA andcontaminant dsRNA (biotin-derivatized (or biotinylated) is furthercontacted with a surface to which streptavidin or avidin is covalentlyattached, thereby binding the biotinylated RNase III, includingbiotinylated RNase III bound to the contaminant dsRNA; upon removal fromthe solution of the surface to which the streptavidin or avidin iscovalently attached, the solution is substantially free, virtually free,essentially free, practically free, extremely free or absolutely free ofdsRNA. Thus, in these embodiments, said purifying the ssRNA or mRNA (orprecursor thereof), comprises contacting the solution comprising the RNAcomposition and the derivatized dsRNA-specific protein (e.g., thebiotinylated RNase III) with a surface to which binds the derivatizeddsRNA-specific protein, and removing the surface from said solution.

In some preferred embodiments wherein the dsRNA-specific protein is a3′-to-5′ exoribonuclease, the method comprises: contacting the ssRNA ormRNA (or precursor thereof) with the 3′-to-5′ exoribonuclease in aTris-buffered (e.g., 20 mM; pH 7.5) solution comprising divalentmagnesium cations (e.g., 5 mM) and a monovalent salt at a concentrationof at least 50 mM (e.g., 150 mM NaCl) and incubating under conditionswherein the exoribonuclease binds to the dsRNA and is enzymaticallyactive, and then cleaning up the mRNA (or precursor thereof) from theexoribonuclease and the other components, including the exoribonucleasedigestion products, to generate treated mRNA (or precursor thereof) thatis substantially free, virtually free, essentially free, practicallyfree, extremely free or absolutely free of dsRNA.

In some embodiments wherein a purification method comprising aseparation device is used to generate at least partially purified ssRNAor mRNA (e.g., a purification method comprising gravity flow or lowpressure chromatography, HPLC or preparative electrophoresis), inaddition to said purification method, the method further comprises(either prior to or after said purification method): contacting thessRNA or mRNA (or precursor thereof) with a dsRNA-specific protein. Insome embodiments, the dsRNA-specific protein is RNase III in a bufferedsolution that contains magnesium cations at a concentration of about 1mM to about 4 mM and a monovalent salt at a concentration of at leastabout 100 mM (preferably, at least about 100-300 mM) to generate treatedmRNA (or precursor thereof) that is substantially free, virtually free,essentially free, practically free, extremely free or absolutely free ofdsRNA. In preferred embodiments, the treated mRNA (or precursor thereof)is at least practically free of dsRNA.

In some other embodiments, the dsRNA-specific protein is adsRNA-specific antibody (e.g., the J2 or K1 antibody from English andScientific Consulting, Szirák, Hungary) in a buffered solution thatcontains a monovalent salt at a concentration of at least about 100 mM(preferably, at least about 100-300 mM), and incubating under conditionswherein the dsRNA-specific antibody binds to the dsRNA, and thencleaning up the mRNA (or precursor thereof) from the dsRNA-specificantibody, at least some of which is bound to the dsRNA, and the othercomponents to generate purified mRNA (or precursor thereof) that issubstantially free, virtually free, essentially free, practically free,extremely free or absolutely free; in some embodiments, thedsRNA-specific antibody is used to assay for the amount of dsRNA presentin the ssRNA (e.g., mRNA or precursor thereof). In some of theseembodiments, the dsRNA-specific antibody can be removed from the mRNA(or precursor thereof) by one of several means (e.g., by usingcommercially available particles such as magnetic particles or beads towhich protein A or protein G is attached to precipitate thedsRNA-specific antibody).

Still further, in some embodiments of any of the above methods, thetreated or purified ssRNA or mRNA (or precursor thereof) is furthercleaned up using the RNA Quick Cleanup Method comprising organic (e.g.,phenol-chloroform) extraction, ammonium acetate precipitation, alcoholprecipitation and/or alcohol washing of the precipitate (e.g., 70%ethanol washing). In some other embodiments, the ssRNA or mRNA (orprecursor thereof) is further cleaned up or purified using a rapid gelfiltration method with a cross-linked dextran (e.g., Sephadex, e.g., aSephadex spin column) in order to separate low molecular weightmolecules, such as salts, buffers, nucleotides and smalloligonucleotides, solvents (e.g., phenol, chloroform) or detergents fromthe ssRNA or mRNA. In some other embodiments, the ssRNA or mRNA ispurified or further purified by chromatography or other means asdescribed elsewhere herein.

For example, in one embodiment, the present invention provides methodsfor synthesizing an in vitro transcribed (IVT) RNA composition, and thencontacting the IVT RNA composition with a dsRNA-specific RNase, such asRNase III, under conditions wherein contaminant dsRNA can bereproducibly digested and ssRNA molecules that do not induce or activatea dsRNA innate immune response pathway or RNA sensor can reliably begenerated.

In some embodiments of the methods, compositions or kits forreprogramming a eukaryotic cell, such as a human or animal cell, thessRNA mRNA (or a precursor to the mRNA, such as IVT-RNA prior to cappingand/or polyadenylation) is purified or treated using at least one methodselected from the group consisting of: (i) a process comprising treatingthe mRNA (or a precursor thereof) with one or more enzymes thatspecifically digest one or more RNA contaminant molecules or contaminantDNA molecules; (ii) chromatography on a gravity flow or HPLC column andan eluant solution that results in removal of contaminant RNA molecules(particularly contaminant dsRNA molecules); and (iii) a processcomprising treating the mRNA (or a precursor thereof) with adsRNA-specific RNase in a reaction mixture under conditions wherein thedsRNA is digested; in some embodiments, the method further comprises:purifying the mRNA from the components of the dsRNA-specific RNasereaction mixture and the dsRNA digestion products. In some preferredembodiments of the method comprising treating the ssRNA or mRNA with adsRNA-specific RNase, the dsRNA-specific RNase is an endoribonuclease(endoRNase). In some preferred embodiments, the endoRNase is RNase III(e.g., E. coli RNase III). In some other embodiments, the dsRNA-specificRNase is an exoribonuclease (exoRNase). In some embodiments, theexoRNase is a protein that exhibits dsRNA-specific 3′-to-5′ exoRNaseactivity.

In some embodiments, the invention also provides a method for making thepurified RNA compositions comprising ssRNA molecules that aresubstantially free, virtually free, essentially free, practically free,extremely free or absolutely free of contaminant dsRNA molecules, themethod comprising: treating in vitro-synthesized RNA comprising one ormore different ssRNA molecules and contaminant dsRNA molecules with adouble-strand-specific RNase in a reaction mixture under conditionswherein the dsRNA is digested, and then purifying the ssRNA moleculesfrom the components of the double-strand-specific RNase reaction mixtureand the dsRNA digestion products. In some embodiments, thedsRNA-specific RNase is RNase III and the reaction mixture comprisesdivalent magnesium cations at a concentration of less than about 5 mM,preferably about 1 mM to about 4 mM, and most preferably about 2 mM toabout 3 mM, or 2 mM. In some embodiments of this method, the ssRNAmolecules are substantially free, virtually free, essentially free,practically free, extremely free or absolutely free of dsRNA contaminantmolecules that activate an RNA sensor or an RNA interference (RNAi)response; in particular embodiments, the RNA sensor is selected from thegroup consisting of RNA-dependent protein kinase (PKR), retinoicacid-inducible gene-I (RIG-I), Toll-like receptor (TLR)3, TLR7, TLR8,melanoma differentiation associated gene-5 protein (MDA5), and2′-5′oligoadenylate synthetase (2′-5′ OAS or OAS). In certainembodiments, the purified RNA compositions or preparations generate nosignificant Toll-Like Receptor (TLR3)-mediated immune response whenintroduced into the cell.

In other embodiments, the iPS cell induction factor is selected from thegroup consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. Inparticular embodiments, the introducing comprises introducing mRNAencoding a plurality of iPS cell induction factors into the somaticcell. In further embodiments, the plurality of iPS cell inductionfactors comprises each of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. Infurther embodiments, the plurality of iPS cell induction factorscomprises OCT4, SOX2, KLF4, LIN28, NANOG, and at least one MYC proteinselected from the group consisting of wild-type c-MYC long, mutantc-MYC(T58A), wild-type c-MYC short and L-MYC. In further embodiments,the plurality of iPS cell induction factors does not comprise LIN28 orNANOG. In preferred embodiments, the MYC protein in the plurality of iPScell induction factors is c-MYC(T58A). In some embodiments, mRNA encodesone or more reprogramming factors or iPS cell induction factors selectedfrom the group consisting of OCT4, SOX2, KLF4, LIN28, NANOG, wild-typec-MYC long, c-MYC(T58A) (Wang X et al., 2011; Wasylishen A R, et al.2011), wild-type c-MYC short and L-MYC. In some embodiments, the mRNAencodes OCT4, SOX2, KLF4, and at least one MYC protein selected from thegroup consisting of wild-type c-MYC long, c-MYC(T58A), wild-type c-MYCshort and L-MYC. In some preferred embodiments, the MYC protein encodedby the mRNA is the c-MYC(T58A). In some other preferred embodiments, theMYC protein encoded by the mRNA is wild-type c-MYC short.

In some other preferred embodiments, the MYC protein encoded by the mRNAis L-MYC. In some embodiments, the mRNA further encodes the NANOGprotein. In some embodiments, the mRNA used for reprogramming human oranimal somatic cell to a dedifferentiated cell or an iPS cell encodesOCT4, SOX2, KLF4, LIN28, NANOG and at least one MYC protein selectedfrom the group consisting of wild-type c-MYC long, c-MYC(T58A),wild-type c-MYC short and L-MYC.

In additional embodiments, the cell is a fibroblast. In otherembodiments, the reprogrammed cell is a pluripotent stem cell. In otherembodiments, the dedifferentiated cell expresses NANOG and TRA-1-60. Infurther embodiments, the cell is in vitro. In additional embodiments,the cell resides in culture. In particular embodiments, the cells residein MEF-conditioned medium. In some preferred embodiments, an RNaseinhibitor (e.g., SCRIPTGUARD™ RNase inhibitor, CELLSCRIPT, INC.,Madison, Wis., USA) is added to the culture medium if the mediumcontains serum, conditioned medium, or a cell extract. In some preferredembodiments, the cell is cultured in medium on an extracellular matrix(e.g., a MATRIGEL™-type matrix) in the absence of a feeder layer. Inother embodiments, the cells reside in a human or animal subject.

In certain embodiments, the present invention provides compositionscomprising an mRNA encoding a reprogramming factor or an iPS cellinduction factor, the mRNA having pseudouridine or1-methyl-pseudouridine in place of uridine. In certain embodimentswherein the mRNA encoding a reprogramming factor or an iPSC inductionfactor comprises pseudouridine or 1-methyl-pseudouridine in place ofuridine, the mRNA also further comprises 5-methylcytidine in place ofcytidine. In other embodiments, the composition comprises mRNA encodinga plurality of iPS cell induction factors, selected from the groupconsisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. In furtherembodiments, the plurality comprises three or more, or four or more, orfive or more, or six iPS cell induction factors.

In certain embodiments, the compositions described above are packaged ina kit. In some embodiments, the compositions comprise a transfectionreagent and an mRNA encoding a reprogramming factor or an iPS cellinduction factor.

In some embodiments, the present invention provides compositions orsystems or kits comprising: a) single-stranded RNA (ssRNA) that encodesa protein, wherein the ssRNA is a product of in vitro transcription of aDNA template by an RNA polymerase; b) a double-stranded RNA (dsRNA)specific endoribonuclease III (endoRNase III) protein (or otherdsRNA-specific protein); and c) magnesium cations present at aconcentration of about 1-4 mM. In particular embodiments, the magnesiumcations are present at a concentration of about 1-3 mM. In certainembodiments, the magnesium ions are present at a concentration betweenabout 1-3 mM (e.g., about 1.0 . . . 1.3 . . . 1.6 . . . 1.9 . . . 2.2 .. . 2.5 . . . 2.8 . . . and 3.0 mM). In particular embodiments, thecompositions and systems further comprise a salt providing an ionicstrength of at least equivalent to 50 mM potassium acetate or potassiumglutamate (e.g., at least 50 mM . . . at least 75 mM . . . at least 100mM . . . at least 150 mM or more). In some embodiments, the ssRNA:exhibits a therapeutic RNA sequence, is an mRNA encoding a therapeuticprotein, is an mRNA encoding a reporter protein, or is an mRNA encodinga cell reprogramming factor.

In particular embodiments, the present invention provides compositionsor systems comprising: a) a ssRNA or mRNA encoding a reprogrammingfactor, and b) magnesium ions present at a concentration of about 1-4 mM(e.g., about 1.0 . . . 1.3 . . . 1.6 . . . 1.9 . . . 2.2 . . . 2.5 . . .2.8 . . . 3.0 . . . 3.4 . . . 3.8 . . . 4.2 . . . 4.8 mM).

In certain embodiments, the dsRNA-specific protein is a dsRNA-specificRNase, an endoribonuclease, or RNase III, or a 3′-to-5′ exoribonuclease.

In some embodiments, the present invention provides methods ofgenerating an RNA preparation (or RNA composition) comprising:contacting in vitro transcribed RNA with a composition comprising a) adouble-stranded RNA-specific (dsRNA-specific) endoribonuclease III(endoRNase III) protein, and b) magnesium cations present at aconcentration of about 1-4 mM; such that an RNA preparation isgenerated.

In certain embodiments, the RNA preparation is practically free,extremely free, absolutely free of dsRNA. In further embodiments, themethods further comprise cleaning up the RNA preparation by removing atleast one of the endoRNase III, or nucleotides, from the RNApreparation. In certain embodiments, the methods further comprise: (i)extracting the RNA preparation with organic solvents (e.g., such asphenol and/or chloroform); (ii) precipitating the in vitro transcribedssRNA with ammonium acetate; and/or (iii) washing the ammonium acetateprecipitate with an alcohol such as 70% ethanol. In particularembodiments, the cleaning up employs a dsRNA-specific antibody. In otherembodiments, the cleaning up further comprises: using an antibody thatbinds to the endoRNase III and/or the dsRNA-specific antibody and thenprecipitating the antibody with magnetic particles or beads to whichprotein A or protein G is attached.

In some embodiments, the present invention provides methods forobtaining translation of at least one protein of interest in a human oranimal cell comprising: repeatedly or continuously introducing into thecell an RNA composition comprising mRNA that encodes the at least oneprotein of interest, wherein the RNA composition has been treated withRNase III, whereby the RNA composition is practically free, extremelyfree or absolutely free of dsRNA (e.g., meaning that less than 0.01%,less than 0.001%, or less than 0.0002%, respectively, of the RNA in thecomposition is dsRNA of a size greater than about 40 basepairs inlength), and culturing the cell under conditions wherein the cellsurvives and grows, and wherein the mRNA is translated. In certainembodiments, cell is ex vivo in culture or in vivo. In furtherembodiments, composition generates substantially no Toll-Like Receptor 3(TLR3) mediated immune response when introduced into or contacted withor injected into a human or animal cell or subject.

In other embodiments, the composition does not generate an innate immuneresponse that is sufficient to cause substantial inhibition of cellularprotein synthesis or dsRNA-induced apoptosis when the treated RNAcomposition is repeatedly introduced into a living human or animal cellor subject. In some embodiments, the cell is a somatic cell, amesenchymal stem cell, a reprogrammed cell, a non-reprogrammed cell, orother type of cell. In particular embodiments, the method is performedwithout the use any exogenous protein (e.g., B18R), siRNA, or smallmolecule agent that inhibits or reduces the activation, induction or theexpression of one or more proteins in an innate immune response pathway.

In certain embodiments, the method further comprises: treating the cellwith a protein, siRNA, mRNA (e.g. encoding B18R or Vaccinia virus E3L,or K3L), or small molecule agent that inhibits or reduces theactivation, induction or expression of one or more RNA sensors orproteins in an innate immune response pathway, wherein the treating isprior to and/or during the introducing.

In some embodiments, the cell exhibits a first differentiated state orphenotype prior to the introducing, and exhibits a second differentiatedstate or phenotype after the introducing.

In some embodiments, the cell, prior to the introducing is anon-reprogrammed cell and after the introducing is a reprogrammed cell,wherein the reprogrammed cell is a dedifferentiated cell, an inducedpluripotent stem cell, a transdifferentiated cell, a differentiated orredifferentiated somatic cell. In further embodiments, the introducingis repeated daily for at least 2 days. In particular embodiments, theintroducing is repeated daily for at least 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17, days, 18 days, 19 days, 20 days, 21 days . .. 30 days . . . 50 days or more.

In some embodiments, the present invention provides compositions orsystem comprising: a) a buffer or other aqueous solution, and b) ssRNAmolecules encoding at least one protein, wherein: i) the at least oneprotein is a reprogramming factor, and/or ii) wherein the ssRNAmolecules contain at least one modified base that reduces the activationof an innate immune response pathway in a cell compared to ssRNAmolecules exhibiting the same sequence but lacking the at least onemodified base, and wherein the composition is practically free ofdouble-stranded RNA molecules.

In certain embodiments, the ssRNA is characterized by at least one (orat least two, or at least three, or at least four, or at least five, orall) of the following: i) encodes a reprogramming factor; ii) encodes aCD protein, meaning a protein identified in the cluster ofdifferentiation system; iii) encodes an enzyme; iv) encodes a protein inthe immunoglobulin super family; v) encodes a cytokine or chemokine; vi)encodes a cell surface receptor protein; vi) encodes a protein in a cellsignaling pathway; vii) encodes an antibody; viii) encodes a T cellreceptor; vix) encodes a protein that reduces or suppresses an innateimmune response comprising interferon (IFN) production or response; x)encodes a reporter protein; xi) contains one or more modified bases;xii) exhibits a cap structure; xiii) exhibits a Cap I structure wherethe 5′ penultimate nucleotide comprises a 2′-O-methyl-ribosyl group;xiv) exhibits a poly A tail; xv) does not contain any modified basesother than a 5′ cap nucleotide, if present; xvi) exhibits at least oneheterologous sequence selected from among: a 5′ UTR sequence, Kozaksequence, an IRES sequence, and 3′ UTR sequence; and xvii) encodes aniPS cell induction factor.

In certain embodiments, the reporter is selected from among Aequoreavictoria jellyfish aequorin; a luciferase (e.g., encoding one luciferaseselected from the group consisting of: Photinus pyralis or NorthAmerican firefly luciferase); Luciola cruciata or Japanese firefly orGenji-botaruluciferase; Luciola italic or Italian firefly luciferase);Luciola lateralis or Japanese firefly or Heike luciferase; Luciolamingrelica or East European firefly luciferase; Photuris pennsylvanicaor Pennsylvania firefly luciferase; Pyrophorus plagiophthalamus or Clickbeetle luciferase; Phrixothrix hirtus or Railroad worm luciferase;Renilla reniformis or wild-type Renilla luciferase; Renilla reniformisRluc8 mutant Renilla luciferase; Renilla reniformis Green Renillaluciferase; Gaussia princeps wild-type Gaussia luciferase; Gaussiaprinceps Gaussia-Dura luciferase; Cypridina noctiluca or Cypridinaluciferase; Cypridina hilgendorfii or Cypridina or Vargula luciferase;Metridia longa or Metridia luciferase; and Oplophorus gracilorostris orOLuc luciferase; or encoding 2 different luciferases selected from thegroup consisting of native Firefly luciferase and Renilla luciferase;Red Firefly luciferase and wild-type Renilla luciferase; Red Fireflyluciferase and Green Renilla luciferase; Gaussia luciferase and Renillaluciferase; Gaussia luciferase and Green Renilla luciferase; Gaussialuciferase and Firefly luciferase; Gaussia luciferase and Red Fireflyluciferase; Gaussia luciferase and Cypridina luciferase; Cypridinaluciferase and Renilla luciferase; Cypridina luciferase and GreenRenilla luciferase; Cypridina luciferase and Red Firefly luciferase; orencoding 3 different luciferases selected from the group consisting of:Cypridina luciferase, Gaussia luciferase, and any Firefly luciferase;and Cypridina luciferase, any Renilla luciferase and Fireflyluciferase); and a fluorescent protein (e.g., encoding a fluorescentprotein selected from the group consisting of: a Phycobiliprotein (e.g.R-Phycoerythrin (R-PE), B-Phycoerythrin (B-PE), C-Phycocyanin (CPC), andAllophycocyanin (APC)); an Aequorea green fluorescent protein; anAequorea blue fluorescent protein (BFP); an Aequorea cyan fluorescentprotein (CFP); an Aequorea yellow fluorescent protein (YFP); an Aequoreaviolet-excitable green fluorescent protein (Sapphire); an Aequoreacyan-excitable enhanced green protein fluorescent protein (EGFP);Discosoma red fluorescent protein; a variant of monomeric Discosoma redfluorescent protein referred to as a Discosoma “mFruits” (m formonomeric) fluorescent protein [e.g. Discosoma yellow fluorescentprotein (mHoneydew); Discosoma blue fluorescent protein (mBlueberry);Discosoma orange fluorescent protein (mOrange)]; Zoanthus yellowfluorescent protein; Obelia green fluorescent proteins; Renillareniformis sea pansy green fluorescent proteins; Anthozoa fluorescentproteins; lancelet fluorescent protein; copepod crustacean fluorescentprotein; Entacmaea quadricolor far-red fluorescent protein; Anemoniasulcata red fluorescent protein; Trachyphyllia geoffroyi “Kaede” redfluorescent protein; Lobophyllia hemprichii fluorescent protein;Dendronephthya fluorescent protein; a Cnidaria fluorescent protein;Arthropoda fluorescent protein; and Chordata fluorescent protein; amonomeric Galaxea fluorescent protein; a monomeric Fungia concinnafluorescent protein; a monomeric Lobophyllia hemprichii fluorescentprotein; a monomeric Pectiniidae fluorescent protein; a monomericDendronephthya fluorescent protein; a monomeric Montipora fluorescentprotein; and a monomeric Clavularia s fluorescent protein).

In particular embodiments, the ssRNA exhibits a cap structurecomprising: i) a cap1 structure, wherein the 2′ hydroxyl of the ribosein the 5′ penultimate nucleotide is methylated, ii) a 5′ cap comprising7-methylguanine, and/or iii) an anti-reverse cap analog (ARCA), or athio-ARCA. In further embodiments, the ssRNA molecule exhibits a poly-Atail composed of at least 50 A residues or at least 100-200 A residues(e.g., at least 50 . . . 75 . . . 100 . . . 150 . . . 200 . . . ormore). In particular embodiments, the 5′ UTR or 3′ UTR exhibited by thessRNA is a sequence exhibited by a Xenopus or human alpha- (α-) globinor beta- (β-) globin mRNA, or wherein the 5′ UTR is a sequence exhibitedby tobacco etch virus (TEV) RNA.

In other embodiments, the ssRNA comprises or consists of at least onemodified ribonucleoside selected from the group consisting ofpseudouridine (Ψ), 1-methyl-pseudouridine (m¹Ψ), 5-methylcytidine (m⁵C),5-methyluridine (m⁵U), 2′-O-methyluridine (Um or m^(2′-O)U),2-thiouridine (s²U), and N⁶-methyladenosine (m⁶A) in place of at least aportion of the corresponding unmodified canonical ribonucleoside. Inparticular embodiments, with the exception of the 5′ cap nucleotide ifpresent, the ssRNA contains only the canonical G, A, C and U nucleicacid bases.

In some embodiments, the ssRNA comprises at least one modifiedribonucleoside, the at least one modified ribonucleoside being selectedfrom the group consisting of: (i) pseudouridine (Ψ),1-methyl-pseudouridine (m¹Ψ), 5-methyluridine (m⁵U), 2′-O-methyluridine(Um or m^(2′-O)U), and 2-thiouridine (s²U) in place of all or almost allof the canonical uridine residues; (ii) 5-methylcytidine (m⁵C) in placeof all or almost all of the canonical cytidine residues; and/or (iii)N⁶-methyladenosine (m⁶A) in place of all or almost all of the canonicaladenosine residues.

In further embodiments, only a portion of a canonical ribonucleoside isreplaced by the corresponding modified ribonucleoside (e.g., wherein aportion means 1-25%, 25-50%, or 50-99% of the canonical ribonucleosideis replaced).

In certain embodiments, the at least one modified ribonucleosidecomprises or consists of pseudouridine (Ψ) or 1-methyl-pseudouridine(m¹Ψ) in place of all or almost all of the canonical uridine residues,and/or 5-methylcytidine (m⁵C) in place of all or almost all of thecanonical cytidine residues.

In other embodiments, only a portion of the canonical uridine residuesare replaced by pseudouridine or 1-methyl-pseudouridine residues and/oronly a portion of the canonical cytidine residues are replaced by5-methylcytidine residues (e.g., wherein a portion means 1-25%, 25-50%,or 50-99% of one or both canonical ribonucleosides are replaced). Incertain embodiments, except with respect to the nucleic acid basescomprising the 5′ cap, the mRNA is composed of (or consists of) onlyunmodified canonical G, A, C and U nucleic acid bases. In otherembodiments, the protein encoded by the ssRNA that reduces or suppressesan innate immune response comprising interferon (IFN) production orresponse is selected from among E3L protein, K3L protein, and B18Rprotein, or a functional fragment or variant of any thereof. In certainembodiments, the composition is practically free, extremely free orabsolutely free of dsRNA.

In some embodiments, the ssRNA encodes at least one protein selectedfrom the group consisting of: MYOD, ASCL1, MYT1L, NEUROD1, POU3F2, OCT4,SOX2, KLF4, LIN28, NANOG, MYC, c-MYC, c-MYC(T58A), L-MYC, ETS2, MESP1GATA4, HAND2, TBX5, MEF2C, ASCL1, EN1, FOXA2, LMX1A, NURR1, PITX3,HNF1a, HNF4a, FOXA1, FOXA2, FOXA3, GATA4, erythropoietin, and a CDprotein; or a functional fragment or variant of any of the preceding.

In further embodiments, the CD protein is selected from: a cell surfacereceptor, a ligand for a cell surface receptor, a cell signalingmolecule, a cell adhesion molecule, a co-stimulating molecule, acomplement system protein, a protein comprising a class I or class IImajor histocompatibility antigen, an inhibitor of a cell signalingmolecule, a transporter of a cell signaling molecule, and an effectormolecule of an innate or adaptive immune response. In other embodiments,the CD protein is selected from: CD1a; CD1b; CD1c; CD1d; CD1e; CD2;CD3d; CD3e; CD3g; CD4; CD5; CD6; CD7; CD8a; CD8b; CD9; CD10; CD11a;CD11b; CD11c; CD11d; CDw12; CD14; CD16a; CD16b; CD18; CD19; CD20; CD21;CD22; CD23; CD24; CD25; CD26; CD27; CD28; CD29; CD30; CD31; CD32; CD33;CD34; CD35; CD36; CD37; CD38; CD39; CD40; CD41; CD42a; CD42b; CD42c;CD42d; CD44; CD45; CD46; CD47; CD48; CD49a; CD49b; CD49c; CD49d; CD49e;CD49f; CD50; CD51; CD52; CD53; CD54; CD55; CD56; CD57; CD58; CD59; CD61;CD62E; CD62L; CD62P; CD63; CD64; CD66a; CD66b; CD66c; CD66d; CD66e;CD66f; CD68; CD69; CD70; CD71; CD72; CD74; CD79a; CD79b; CD80; CD81;CD82; CD83; CD84; CD85a; CD85c; CD85d; CD85e; CD85f; CD85g; CD85h;CD85i; CD85j; CD85k; CD86; CD87; CD88; CD89; CD90; CD91; CD92; CD93;CD94; CD95; CD96; CD97; CD98; CD99; CD100; CD101; CD102; CD103; CD104;CD105; CD106; CD107a; CD107b; CD108; CD109; CD110; CD111; CD112; CD113;CD114; CD115; CD116; CD117; CD118; CD119; CD120a; CD120b; CD121a;CD121b; CD122; CD123; CD124; CD125; CD126; CD127; CD129; CD130; CD131;CD132; CD133; CD134; CD135; CD136; CD137; CD138; CD139; CD140a; CD140b;CD141; CD142; CD143; CD144; CD146; CD147; CD148; CD150; CD151; CD152;CD153; CD154; CD155; CD156a; CD156b; CD157; CD158a; CD158b1; CD158b2;CD158c; CD158d; CD158e; CD158f1; CD158g; CD158h; CD158i; CD158j; CD158k;CD158z; CD159a; CD159c; CD160; CD161; CD162; CD163; CD163b; CD164;CD165; CD166; CD167a; CD167b; CD168; CD169; CD170; CD171; CD172a;CD172b; CD172g; CD173; CD177; CD178; CD179a; CD179b; CD180; CD181;CD182; CD183; CD184; CD185; CD186; CD191; CD192; CD193; CD194; CD195;CD196; CD197; CDw198; CDw199; CD200; CD201; CD202b; CD203a; CD203c;CD204; CD205; CD206; CD207; CD208; CD209; CD210; CDw210b; CD212;CD213a1; CD213a2; CD214; CD215; CD217; CD218a; CD218b; CD220; CD221;CD222; CD223; CD224; CD225; CD227; CD228; CD229; CD230; CD231; CD232;CD233; CD234; CD235a; CD235b; CD236; CD238; CD239; CD240CE; CD240D;CD241; CD242; CD243; CD244; CD245; CD246; CD247; CD248; CD249; CD252;CD253; CD254; CD256; CD257; CD258; CD261; CD262; CD263; CD264; CD265;CD266; CD267; CD268; CD269; CD270; CD271; CD272; CD273; CD274; CD275;CD276; CD277; CD278; CD279; CD280; CD281; CD282; CD283; CD284; CD286;CD288; CD289; CD290; CD292; CDw293; CD294; CD295; CD296; CD297; CD298;CD299; CD300a; CD300b; CD300c; CD300d; CD300e; CD300f; CD300g; CD301;CD302; CD303; CD304; CD305; CD306; CD307a; CD307b; CD307c; CD307d;CD307e; CD309; CD312; CD314; CD315; CD316; CD317; CD318; CD319; CD320;CD321; CD322; CD324; CD325; CD326; CD327; CD328; CD329; CD331; CD332;CD333; CD334; CD335; CD336; CD337; CD338; CD339; CD340; CD344; CD349;CD350; CD351; CD352; CD353; CD354; CD355; CD357; CD358; CD360; CD361;CD362; and CD363; or a functional fragment or variant of any of thepreceding.

In further embodiments, the in vitro-transcribed ssRNA encodes aplurality of reprogramming factors. In further embodiments, the RNApreparation generates substantially no Toll-Like Receptor 3 (TLR3)mediated immune response when introduced into or contacted with orinjected into a human or animal cell or subject. In additionalembodiments, the RNA preparation does not generate an innate immuneresponse that is sufficient to cause substantial inhibition of cellularprotein synthesis or dsRNA-induced apoptosis when the treated RNAcomposition is repeatedly introduced into a living human or animal cellor subject.

In some embodiments, the present invention provides methods of making anRNA preparation comprising: a) processing in vitro transcribed RNA by:i) exposure to a dsRNA-specific endoribonuclease III protein in areaction mixture comprising a salt that results in an ionic strength atleast as high as potassium acetate at a concentration of about 50-300 mMand a final magnesium concentration of about 1-4 mM, and/or ii) passagethrough a chromatographic or electrophoretic separation device; whereinthe processing the in vitro transcribed RNA generates an RNA preparationthat is practically free, extremely free or absolutely free ofdouble-stranded RNA, and wherein the in vitro transcribed RNA encodes atleast one protein, wherein: i) the at least one protein is areprogramming factor, and/or ii) wherein the in vitro transcribed RNAcontains at least one modified base that reduces the induction oractivation of an RNA sensor or innate immune response pathway in a cell.

In particular embodiments, the chromatographic separation device is agravity flow or HPLC column. In certain embodiments, the presentinvention provides methods of making an RNA preparation comprising: a)contacting a composition containing single-stranded RNA (ssRNA) anddouble-stranded RNA (dsRNA) with a solution that contains RNase III anda monovalent salt at a concentration of at least 50 mM, but which lacksdivalent magnesium cations, such that a mixture is generated, b)incubating the mixture under conditions such that the RNase III binds tothe dsRNA but is not generally enzymatically active, and c) cleaning upthe ssRNA from the RNase III, at least some of which is bound to thedsRNA, to generate an RNA preparation that contains ssRNA and issubstantially free, virtually free, essentially free, or practicallyfree of dsRNA (e.g., meaning, respectively, that less than: 0.5%, 0.1%,0.05%, 0.01%, 0.001% or 0.0002% of the mass of the RNA in the treatedssRNA composition is dsRNA of a size greater than about 40 basepairs).

In some embodiments, the present invention provides methods of obtainingexpression of at least one protein of interest in a cell comprising:contacting a cell with an RNA composition comprising invitro-synthesized ssRNA that encode at least one protein of interestsuch that the at least one protein of interest is expressed in the cell,wherein the contacting: a) is conducted at least once daily for aplurality of days, or b) is conducted a plurality of time over at least24 hours; and wherein the contacting does not induce an innate immuneresponse that: i) kills the cell; ii) is sufficient to inhibit proteinsynthesis by two-fold or greater; and/or iii) induces or activatesproteins involved in an apoptosis pathway.

In certain embodiments, the at least one protein of interest is areprogramming factor, and wherein the plurality days is sufficientnumber of days to reprogram the cell. In certain embodiments, theplurality of days is at least 2 days, at least 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17, days, 18 days, 19 days, 20 days, 21 days . .. 30 days . . . 50 days or more. In particular embodiments, the ssRNAcomprises at least one of the following: a 5′ cap, a 5′ untranslatedregion, a 5′ Kozak sequence, a 3′ untranslated region, and a poly(A)tail. In further embodiments, the composition is at least practicallyfree of double stranded RNA. In further embodiments, the cell is locatedin a subject or is located ex vivo in culture. In some embodiments, thecomposition is free of a protein, siRNA, or small molecule agent thatinhibits or reduces the activation, induction or expression of one ormore RNA sensors or proteins in an innate immune response pathway.

In certain embodiments, the cell is present in a culture medium, whereinthe culture medium: i) is free of feeder cells, and/or ii) comprises atleast one reagent selected from the group consisting of: a TGF-betainhibitor and a MEK inhibitor. In other embodiments, the cell is presentin a culture medium that lacks a biological substrate.

In some embodiments, the RNA molecule is a therapeutic RNA sequence, anmRNA encoding a therapeutic protein, an mRNA encoding a reporterprotein, or an mRNA encoding a cell reprogramming factor.

In certain embodiments, the composition comprises at least oneadditional component selected from: i) a monovalent salt at aconcentration of at least 50 mM; ii) a cell; iii) a protein, siRNA, orsmall molecule agent that inhibits or reduces the activation, inductionor expression of one or more RNA sensors or proteins in an innate immuneresponse pathway; and iv) a dsRNA binding protein. In some embodiments,the cell is a somatic cell, a mesenchymal stem cell, a reprogrammedcell, a non-reprogrammed cell, In particular embodiments, prior to thecontacting, the composition is treated with a dsRNA-specific RNase suchthat substantially or practically all contaminant dsRNA is digested.

In particular embodiments, the cell before the contacting for aplurality of days exhibits a first differentiated state or phenotype,and after the contacting for a plurality of days, exhibit a seconddifferentiated state or phenotype.

In particular embodiments, the present invention provides methods formaking ssRNAs for use in reprogramming eukaryotic cells that exhibit afirst differentiated state or phenotype to cells that exhibit a seconddifferentiated state or phenotype by introducing the ssRNAs into thecells at least three times over a period of at least two days, themethod comprising: (i) synthesizing one or more ssRNAs by in vitrotranscription, each of which encodes a reprogramming factor; and (ii)treating the ssRNAs from step (i) with RNase III in a buffered solutionhaving a pH of about 7 to about 9, a monovalent salt having at aconcentration of about 100 mM or higher, divalent magnesium cations at aconcentration of about 1 mM to less than 10 mM for sufficient time andunder conditions wherein dsRNA is digested and ssRNAs that aresubstantially free of dsRNA are generated; in other embodiments, saidintroducing is for at least about: three days, . . . 6 days, . . . 8days, . . . 10 days, . . . 15 days, . . . 18 days, . . . 21 days, . . .28 days, . . . 35 days, . . . 42 days, . . . 50 days, . . . or greaterthan 50 days.

In some embodiments, the present invention provides compositions, kits,or systems comprising: a) a cell and/or RNA encoding at least oneprotein, wherein: i) the at least one protein is a reprogramming factor,and/or ii) wherein the RNA contains at least one modified base thatreduces the activation of an innate immune response pathway in the cell;and b) a culture medium, wherein the culture medium: i) comprises atleast one reagent selected from the group consisting of: a TGF-betainhibitor and a MEK inhibitor; and/or ii) comprises a biologicalsubstrate for the cell, and is free of feeder cells; and/or iii) doesnot comprise either an extracellular matrix or other biologicalsubstrate or feeder cells. In some embodiments, wherein the culturemedium does not comprise either an extracellular matrix or otherbiological substrate or feeder cells, the culture plate or vesselexhibits a treated surface on which the cells adhere and grow as aconfluent layer.

In certain embodiments, the composition or system comprises both thecell and the RNA, wherein the RNA are present inside the cell. Inparticular embodiments, the cell is a reprogrammed cell. In certainembodiments, the reprogrammed cell is a dedifferentiated cell, aninduced pluripotent stem, or a transdifferentiated cell. In someembodiments, the biological substrate comprises vitronectin proteinand/or the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm(EHS) mouse sarcoma cells.

In additional embodiments, the present invention provides methods ofculturing a cell comprising: culturing cells on the culture mediumdescribed above or herein, wherein the cells comprise the RNA describedherein. In some embodiments, the cells exhibit a first differentiatedstate or phenotype prior to the culturing, and exhibit a seconddifferentiated state or phenotype after the culturing. In furtherembodiments, the cells, prior to the culturing, are non-reprogrammedcells and after the culturing are reprogrammed cells, wherein thereprogrammed cells are dedifferentiated cells, induced pluripotent stemcells, transdifferentiated cells, differentiated or redifferentiatedsomatic cells. In particular embodiments, the culturing is continued forat least 2 days, 3 days, . . . 10 days . . . 20 days, or more, or for10-18 days or for about 2-25 days.

In certain embodiments, the present invention provides compositions,kits, and systems comprising: a mixture of mRNAs encoding iPSCreprogramming factors comprising KLF4 (K), MYC (M), OCT4 (0), andSOX2(S), wherein the molar concentration of mRNA encoding O is about3-times higher than the molar concentration of mRNA encoding M and S andwherein mRNA encoding K is between about 1 time and about 3 times themolar concentration of M and S, wherein the RNA composition ispractically free, extremely free or absolutely free of dsRNA. Inparticular embodiments, the mRNAs further encode either LIN28 (L) orNANOG (N) or both, wherein the molar concentration of mRNA encoding L orN, if present, is the same or about the same as the molar concentrationof M and S.

In some embodiments, the present invention provides compositions andsystems comprising: a) a first mixture of different RNA moleculesencoding ten different combinations of the following proteins: KLF4 orfunctional fragment or variant thereof (K), MYC or functional fragmentor variant thereof(M), OCT4 or functional fragment or variantthereof(0), SOX2 or functional fragment or variant thereof (S), LIN28 orfunctional fragment or variant thereof (L), and NANOG or functionalfragment or variant thereof (N), wherein the different RNA molecules arepresent in the composition or system in an approximate molar ratioselected from the group consisting of: KMO_(2.5-3.5)SLN; KMO_(2.5-3.5)S;KMO_(2.5-3.5)SL; K_(1.5-2.5)MO_(2.5-3.5)SLN; K_(2.5-3.5)MO_(2.5-3.5)SLN;K_(1.5-2.5)MO_(2.5-3.5)SL; K_(2.5-3.5)MO_(2.5-3.5)SL;K_(1.5-2.5)MO_(2.5-3.5)S; K_(2.5-3.5)MO_(2.5-3.5)S; or K_(1.5-10.0)LMS;and/or b) a second mixture of different RNA molecules encoding KLF4,c-MYC, OCT4, and SOX2, wherein no other reprogramming genes are presentin the composition or system.

In particular embodiments, no other reprogramming RNA sequences arepresent in the composition or system than recited in the ten differentcombinations. In particular embodiments, the compositions furthercomprise a cell. In certain embodiments, the cell is a reprogrammedcell. In further embodiments, MYC is c-MYC, L-MYC, or c-MYC(T58A). Inadditional embodiments, the approximate molar ratios are selected from:KMO₃SLN; KMO₃S; KMO₃SL; K₂MO₃SLN; K₃MO₃SLN; K₂MO₃SL; K₃MO₃SL; K₂MO₃S;K₃MO₃S; or K_(1.5-2.5)LMS.

In certain embodiments, the present invention provides methods forchanging or reprogramming the state of differentiation or differentiatedstate or phenotype of a cell comprising: introducing a plurality ofdifferent RNA molecules into a cell, wherein the cells exhibits a firstdifferentiated state or phenotype prior to the introducing and exhibitsa second differentiated state or phenotype after the introducing, andwherein the introducing results in an approximate molar ratio of thedifferent RNA molecules in the cell selected from the group consistingof: KMO_(2.5-3.5)SLN; KMO_(2.5-3.5)S; KMO_(2.5-3.5)SL;K_(1.5-2.5)MO_(2.5-3.5)SLN; K_(2.5-3.5)MO_(2.5-3.5)SLN;K_(1.5-2.5)MO_(2.5-3.5)SL; K_(2.5-3.5)MO_(2.5-3.5)SL;K_(1.5-2.5)MO_(2.5-3.5)S; K_(2.5-3.5)MO_(2.5-3.5)S; or K_(1.5-10.0)LMS;wherein K is KLF4 or functional fragment thereof, M is MYC or afunctional fragment thereof, 0 is OCT4 or functional fragment thereof, Sis SOX2 or functional fragment thereof, L is LIN28 or functionalfragment thereof, and N is NANOG or a functional fragment thereof. Inparticular embodiments, the MYC is c-MYC, L-MYC, or c-MYC(T58A).

In other embodiments, the present invention provides methods forchanging or reprogramming the state of differentiation or differentiatedstate or phenotype of a cell comprising: introducing into a cell thatexhibits a first differentiated state or phenotype: i) a first mRNAencoding KLF4, or functional fragment thereof, ii) a second mRNAencoding c-MYC, or functional fragment thereof, iii) a third mRNAencoding OCT-4, or functional fragment thereof, and iv) a fourth mRNAencoding SOX2, or functional fragment thereof, wherein the introducinggenerates a reprogrammed cell that exhibits a second differentiatedstate or phenotype, and wherein no other reprogramming factors, besidesthe first, second, third, and fourth mRNAs are used to reprogram thecell.

In certain embodiments, the present invention provides methods forchanging or reprogramming the state of differentiation or differentiatedstate or phenotype of a cell comprising: introducing into a cell thatexhibits a first differentiated state or phenotype an RNA moleculeencoding c-MYC (T58A) such that a reprogrammed cell that exhibits asecond differentiated state or phenotype is generated.

In some embodiments, the present invention provides methods for reducingor eliminating a symptom or disease of a eukaryotic subject thatexhibits a disease condition, comprising: administering to the subjectan effective dose of an RNA composition comprising ssRNA that encode atleast one therapeutic protein, wherein the RNA composition is at leastsubstantially free, virtually free, essentially free, or practicallyfree of contaminant dsRNA, whereby the symptom or disease is reduced oreliminated. In some embodiments, the RNA composition is practicallyfree, extremely free or absolutely free of dsRNA. In furtherembodiments, the RNA composition does not generate an innate immuneresponse in the subject that is sufficient to cause substantialinhibition of cellular protein synthesis or dsRNA-induced apoptosis whenthe RNA composition is repeatedly or continuously administered to thesubject. In some embodiments, the therapeutic protein is erythropoietinor truncated or mutated version thereof. In certain embodiments, theadministering is conducted at least once per days for at least two days.In some embodiments, the administering is conducted at least daily atleast 1-7 times per week for at least 1 week (e.g., at least 1 week, 2weeks, 3 weeks, 4 weeks, . . . 10 weeks . . . 52 weeks or more).

In other embodiments, the administering is conducted daily or twice perday, with the administering occurring about 1 time per week, 2 times perweek, 3 times per week, 4 times per week, 5 times per week, 6 times perweek, or daily for a period of weeks, months or years.

In some embodiments, the present invention provides compositions orsystems comprising: a) a reprogrammed or differentiated myoblast cell,wherein the myoblast cell comprises an exogenous RNA molecule encodingMYOD protein or functional fragment thereof, and/or b) a reprogrammed ortransdifferentiated neuron cells, wherein the neuron cell comprisesexogenous RNA molecules encoding at least one protein selected from thegroup consisting of: ASCL1 or functional fragment thereof, MYT1L orfunctional fragment thereof, NEUROD1 or functional fragment thereof, andPOU3F2 or functional fragment thereof.

In certain embodiments, the present invention provides methods forreprogramming a non-myoblast cell to a myoblast cell comprising: a)daily, for at least two days, introducing into a non-myoblast cell acomposition comprising in vitro-synthesized ssRNA or mRNA encoding MYODprotein or functional fragment or variant thereof, wherein thecomposition is at least practically free of dsRNA, and b) culturingunder conditions wherein at least a portion of the non-myoblast cellsare reprogrammed or differentiated into myoblast cells.

In particular embodiments, the present invention provides methods forreprogramming non-neuron somatic cells to neuron cells comprising: a)daily, for multiple days, introducing into non-neuron somatic cells acomposition comprising in vitro-synthesized ssRNA or mRNA encoding atleast one protein selected from the group consisting of: ASCL1 orfunctional fragment thereof, MYT1L or functional fragment thereof,NEUROD1 or functional fragment thereof, and POU3F2 or functionalfragment thereof, wherein the composition is practically free, extremelyfree, or absolutely free of dsRNA, and b) culturing under conditionswherein at least a portion of the non-neuron somatic cells arereprogrammed or transdifferentiated into neuron cells. In certainembodiments, the introducing is conducted at least once daily for atleast two days, three days . . . 10 days . . . 365 days, or more.

In some embodiments the present invention provides methods comprisingcontacting a plurality of cultured cells with a total daily dose (and nomore than the total daily dose) of a composition comprising ssRNAsencoding at least one reprogramming factor, wherein said contacting isrepeated for a sufficient number of days such that at least a portion ofsaid plurality of cultured cells are reprogrammed from a firstdifferentiated state or phenotype to a second differentiated state orphenotype, wherein said total daily dose is between about 0.1 microgramand about 1.2 micrograms of said ssRNAs per 10,000 to 100,000 initiallyplated cells (e.g., per 2 mls of culture medium). In some embodiments,the total daily dose is administered once per day. In some embodiments,the total daily dose is administered as two doses per 24 hours . . . 4doses per 24 hours, 8 doses per 24 hours. In some embodiments, themixture of ssRNAs encoding reprogramming factors are introducedcontinuously (e.g., into the culture medium) using a robotic ormicrofluidic device for said introducing. In some embodiments, mixtureof ssRNAs encoding reprogramming factors are introduced continuously(e.g., into the culture medium) and the composition of the proteinreprogramming factors encoded by the mRNA mixture is varied over time.In particular embodiments, the total daily dose is about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, or about 1.2 micrograms of saidssRNA.

In some embodiments, the present invention provides compositions orsystems comprising: a) a buffer or other aqueous solution, and b) RNAmolecules encoding at least one protein, wherein: i) said at least oneprotein is a reprogramming factor, and/or ii) wherein said RNA moleculescontain at least one modified base that reduces the activation of aninnate immune response pathway in a cell, and wherein said compositionis free of double-stranded RNA molecules to a level provided by HPLCpurification, and wherein said composition would generate no detectableToll-Like Receptor 3 (TLR3) mediated immune response when introducedinto or contacted with or injected into a human or animal cell orsubject.

DESCRIPTION OF THE FIGURES

The following FIGURES form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these FIGURES in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a schematic diagram depicting construction, annealing andRNase digestion III of an RNA substrate comprising a 1671-bp dsRNAregion flanked by a 255-base and 136-base 3′-terminal ssRNA tails. Asshown in FIG. 1, correct digestion of this RNA substrate by adsRNA-specific endoRNase, such as RNase III, would be expected to resultin complete digestion of the central 1671-bp dsRNA portion, whileleaving ssRNA tails of 136 bases and 255 bases intact.

FIG. 2 shows that the ability of RNase III to digest dsRNA whilemaintaining the integrity of ssRNA varies based on the concentration ofdivalent magnesium cations in the reaction. The electrophoresis geldepicts digestion of one microgram of the RNA substrate shown in FIG. 1by RNase III at a concentration of 20 nM in a reaction mixturecontaining 33 mM Tris-acetate, pH8, 200 mM potassium acetate anddifferent concentrations of magnesium acetate (Mg(OAc)₂). Lane M) RNAmillennium markers (0.5 kb, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 9 kb); Lane1): No RNase III control with the intact RNA substrate; Lanes 2)-15):RNase III+Mg(OAc)₂ at: 2) 0 mM; 3) 0.1 mM; 4) 0.25 mM; 5) 0.5 mM; 6) 1mM; 7) 2 mM; 8) 3 mM; 9) 4 mM; 10) 5 mM; 11) 6 mM; 12) 7 mM; 13) 8 mM;14) 9 mM; and 15) 10 mM.

FIG. 3 shows that digestion of different starting amounts of Luc2 dsRNAby RNase III, as detected on dot blots using the dsRNA-specificmonoclonal Antibody J2, varies with the [Mg²⁺] used for RNase IIItreatment. Row: 1) Poly I:C; 2) LIN28 dsRNA; 3) Luc2 dsRNA minus RNaseIII plus 1.0 mM Mg(OAc)₂; Rows 4)-17) depict Luc2 dsRNA plus RNase IIIplus Mg(OAc)₂ at: 4) 0 mM; 5) 0.1 mM; 6) 0.25 mM; 7) 0.5 mM; 8) 1 mM; 9)2 mM; 10) 3 mM; 11) 4 mM; 12) 5 mM; 13) 6 mM; 14) 7 mM; 15) 8 mM; 16) 9mM; 17) 10 mM; Row: 18) cMYC mRNA plus RNase III plus 1 mM Mg(OAc)₂.

FIG. 4 shows that digestion of different starting amounts of Luc2 dsRNAby RNase III, as detected on dot blots using the dsRNA-specificmonoclonal Antibody K1, also varies with the [Mg²⁺] used for RNase IIItreatment. Row: 1) Poly I:C; 2) LIN28 dsRNA; 3) Luc2 dsRNA minus RNaseIII plus 1.0 mM Mg(OAc)₂; Rows 4)-17) depict Luc2 dsRNA plus RNase IIIplus Mg(OAc)₂ at: 4) 0 mM; 5) 0.1 mM; 6) 0.25 mM; 7) 0.5 mM; 8) 1 mM; 9)2 mM; 10) 3 mM; 11) 4 mM; 12) 5 mM; 13) 6 mM; 14) 7 mM; 15) 8 mM; 16) 9mM; 17) 10 mM; and Row: 18) cMYC mRNA plus RNase III plus 1 mM Mg(OAc)₂.

FIG. 5 shows that RNase III treatment can effectively digest dsRNAwithout affecting the integrity of either small (255-nt and 156-nt) orlarge (955-nt) ssRNA present in the same composition. Theelectrophoresis gel shows RNase III digestion of a mixture of the RNAsubstrate comprising a 1671-bp dsRNA region and 255-base and 136-basessRNA tails and a 955-nucleotide ssRNA substrate in the presence ofdifferent concentrations of Mg(OAc)₂. Lanes M) RNA millennium markers(0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 9 Kb); Lanes 1)-13) RNase III inthe presence of Mg(OAc)₂ at: 1) 0 mM; 2) 0.1 mM; 3) 0.25 mM; 4) 1 mM; 5)2 mM; 6) 3 mM; 7) 4 mM; 8) 5 mM; 9) 6 mM; 10) 7 mM; 11) 8 mM; 12) 9 mM;and 13) 10 mM.

FIG. 6 shows an analyses performed on the effects of differentconcentrations of Mg(OAc)₂ on completeness of dsRNA digestion andintegrity of ssRNA when the RNase III treatment was performed using 200mM potassium glutamate as a monovalent salt. This is an example of onetype of analysis which was also performed with other monovalent saltsThe electrophoresis gel shows RNase III digestion of a mixture of theRNA substrate comprising a 1671-bp dsRNA region and 255-base and136-base ssRNA tails and a 955-nucleotide ssRNA substrate in thepresence of different concentrations of Mg(OAc)₂. Lane M) RNA millenniummarkers (0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 9 Kb); Lane 1) No-RNaseIII control with standard pH 8 Tris-OAc buffer+KOAc salt; Lane 2) RNaseIII in standard pH 8 Tris-OAc buffer+1 mM Mg(OAc)₂+KOAc salt; Lanes3)-16) RNase III in standard pH 8 Tris-OAc buffer+200 mM Kglutamate saltin the presence of Mg(OAc)₂ at: 3) 0 mM; 4) 0.1 mM; 5) 0.25 mM; 6) 0.5mM; 7) 1 mM; 8) 2 mM; 9) 3 mM; 10) 4 mM; 11) 5 mM; 12) 6 mM; 13) 7 mM;14) 8 mM; 15) 9 mM; and 16) 10 mM.

FIG. 7 shows the activity of RNase III on a mixture of both dsRNA andssRNA substrates in the presence of 1 mM Mg(OAc)₂ and differentconcentrations of potassium glutamate as the monovalent salt. Lane M)RNA millennium markers (0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 9 Kb);Lane 1) 20 nM RNase III in standard pH 8 Tris-OAc buffer+1 mMMg(OAc)₂+200 mM KOAc salt; Lane 2) No-RNase III control with standard pH8 Tris-OAc buffer+1 mM Mg(OAc)₂+200 mM KOAc salt; Lanes 3)-9) RNase IIIin standard pH 8 Tris-OAc buffer+1 mM Mg(OAc)₂+Kglutamate salt at: 3) 0mM; 4) 50 mM; 5) 100 mM; 6) 150 mM; 7) 200 mM; 8) 250 mM; and 9) 300 mM.

FIG. 8 shows the activity of RNase III in separate reactions containingeither a dsRNA substrate (lanes 1-8) or a ssRNA substrate (lanes 10-17)in the presence of 1 mM Mg(OAc)₂ and different concentrations ofpotassium acetate (KOAc) salt. Lanes M) and 9) RNA millennium markers(0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 9 Kb); Lane 1) dsRNA substrate inno-RNase III control in standard pH 8 Tris-OAc buffer+1 mM Mg(OAc)₂+200mM KOAc salt; Lanes 2)-8) dsRNA substrate+RNase III in standard pH 8Tris-OAc buffer+1 mM Mg(OAc)₂+KOAc salt at: 2) 0 mM; 3) 50 mM; 4) 100mM; 5) 150 mM; 6) 200 mM; 7) 250 mM; and 8) 300 mM; Lane 10) ssRNAsubstrate in no-RNase III control in standard pH 8 Tris-OAc buffer+1 mMMg(OAc)₂+200 mM KOAc salt; Lanes 11)-17) ssRNA substrate+RNase III instandard pH 8 Tris-OAc buffer+1 mM Mg(OAc)₂+KOAc salt at: 11) 0 mM; 12)50 mM; 13) 100 mM; 14) 150 mM; 15) 200 mM; 16) 250 mM; and 17) 300 mM.

FIG. 9 shows the completeness of digestion of a dsRNA substrate by RNaseIII treatment in a reaction mixture consisting of 20 nM RNase III in 33mM Tris-OAc buffer, pH 8, with 200 mM KOAc as the monovalent salt and 1mM Mg(OAc)₂ for 10 minutes at 37° C., when the amount of dsRNA wasvaried from 1 microgram up to 20 micrograms. Lane M) RNA millenniummarkers (0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 9 Kb); Lane 1) 1 microgramdsRNA substrate in no-RNase III control in standard Tris-OAc buffer, pH8+1 mM Mg(OAc)₂+200 mM KOAc salt; Lanes 2)-8)+RNase III and dsRNA at: 2)1 microgram; 3) 2 micrograms; 4) 4 micrograms; 5) 8 micrograms; 6) 12micrograms; 7) 16 micrograms; and 8) 20 micrograms.

FIG. 10 shows that firefly luciferase mRNA subjected to the RNase IIItreatment in the presence of 2 mM Mg(OAc)₂ for 30 minutes exhibitedseveral-fold higher levels of in vivo translation when transfected intoBJ fibroblasts compared to the same mRNA subjected to the RNase IIItreatment in the presence of 10 mM Mg(OAc)₂ for 30 minutes. FollowingRNase III treatment, the firefly luciferase mRNA was cleaned up usingthe RNA Quick Cleanup method as described herein and transfected into BJfibroblast cells in triplicate wells. 18 hours post-transfection, thecells were lysed and assayed for the amount of luciferase activityproduced. The amount of luciferase activity (measured in relative lightunits, RLU) was averaged for duplicate assays of the triplicate samples(n=6) and was normalized by the amount of protein in the cell lysate.

FIG. 11 shows a phase contrast image of an iPSC colony reprogrammed inEXAMPLE 11 from a human BJ fibroblast without use of a feeder layer andwithout using B18R protein or any other inhibitor or agent that reducesthe expression of an innate immune response pathway. The iPSC colonywithin a confluent layer of BJ fibroblast cells is shown after 18 daysof transfection with mRNA iPSC induction factors encoding: OCT4, SOX2,KLF4, LIN28, and cMYC(T58A) proteins.

FIG. 12 shows an example of alkaline phosphatase-stained candidate iPScells generated from human BJ fibroblasts using a method of theinvention wherein the BJ fibroblasts were transfected and cultured infeeder-free wells coated with a MATRIGEL™ GFR matrix in mediumcomprising: A) the Feeder-free Reprogramming Medium in EXAMPLE 11 of thepresent invention without LIF protein or, TGFβ or MEK small moleculeinhibitors; B) the Feeder-free Reprogramming Medium in EXAMPLE 11 of thepresent invention with LIF protein and the small molecule inhibitors,SB431542 TGFβ inhibitor and PD0325901 MEK Inhibitor; and C) a PLURITON™commercial reprogramming medium without further addition of LIF or anysmall molecule inhibitors. Examples of positive staining colonies areindicated by the arrows. (Note: in later experiments, we found that themethod for reprogramming of cells that exhibited a first differentiatedstate or phenotype comprising human fibroblasts to cells that exhibiteda second differentiated state or phenotype comprising iPSCs could beperformed in the absence of feeder cells (i.e., feeder-freereprogramming) if the method further comprises the step of adding a TGFβsmall molecule inhibitor and/or a MEK small molecule inhibitor (e.g.,TGFβ inhibitor SB431542 and MEK Inhibitor PD0325901) to the mediumduring the steps of said repeatedly or continuously introducing of theRNA composition comprising ssRNA or mRNA encoding the reprogrammingfactors (e.g., iPSC reprogramming factors); in these embodiments, it wasnot necessary to add LIF protein.

FIG. 13 shows that cells originating from an iPSC colony that werereprogrammed in EXAMPLE 11 from human BJ fibroblasts in the absence offeeder cells stain positive for the pluripotency markers OCT4, NANOG,SSEA4, SOX2, and TRA-1-60. In this embodiment, iPSCs were induced in theabsence of feeder cells, but mRNA encoding B18R protein was alsotransfected into the BJ fibroblasts at the same time as the iPSCreprogramming factor mRNAs.

FIG. 14 shows that RNase III treatment of RNA greatly reduces the levelsof dsRNA detectable by the JS antibody. All the RNAs shown were maudeusing ψTP in place of UTP.

FIG. 15 shows that BJ fibroblasts transfected for 18 straight days withmRNA reprogramming factors expressed the stem cell marker Tra-1-60. BJfibroblasts were transfected with the five factors (5F) 3:1:1:1:1 molarratio of (OCT4, SOX2, KLF4, LIN28 and c-MYC or c-MYC (T58A) RNaseIIItreated, 5mC/ΨTP at a total dose 1.2 μg of mRNA per transfection for 18days. B18R was used at 200 ng/ml in some of the treatments.

FIG. 16 shows that iPSC colonies reprogrammed from human BJ fibroblastsusing mRNA reprogramming factors show stable expression of stem cellmarkers. iPSC colonies were manually picked and passaged five times onNuff feeder layers in iPSC media containing 100 ng/nl of hFGF2. The iPSCcolonies were fixed and processed for immunoflourescence with antibodiesthat recognize stem cell markers OCT4, SOX2 and NANOG.

FIG. 17 A-C shows that iPSC clonal colonies generated by reprogrammingof human BJ fibroblasts to iPS cells using mRNA reprogramming factorsencoding the iPSC induction factors that were picked and cloneddifferentiated into all three germ layers. The iPSC colonies werepassaged 7 times and allowed to differentiate in an embryoid bodyspontaneous differentiation protocol. The differentiated cells expressedmarkers of endoderm (AFP and SOX17), mesoderm (SMA and Desmin), andectoderm (class III beta-tubulin, also known as βIII-tubulin) after theywere fixed and processed for immunofluorescence with antibodies thatrecognized those markers. FIG. 17A shows the results for clone 2, FIG.17B shows the results from clone 3, and FIG. 17C shows the results fromclone 4.

FIG. 18 shows that iPSC colonies that were obtained by reprogramming ofhuman BJ fibroblasts to iPS cells using mRNA reprogramming factorsencoding the iPSC induction factors were stained by alkalinephosphatase, a commonly used embryonic stem cell marker (Takahashi andYamanaka, 2006).

FIG. 19 shows that mRNA encoding L-MYC can substitute for c-MYC forreprogramming human BJ fibroblasts to iPSC cells. The BJ fibroblastswere transfected with RNase III-treated Ψ-mRNA or Ψ- and m5C-mRNAencoding OCT4, SOX2, KLF4, LIN28 and L-MYC for 17 days.

FIG. 20 shows examples of iPSC colonies generated from BJ fibroblastsafter 17 daily transfections with RNase III-treated Ψ-mRNA or Ψ- andm⁵C-mRNA encoding OCT4, SOX2, KLF4, LIN28 and L-MYC for 17 days.Examples of iPSC colonies observed on day 17 are shown at 10× (top 6images with scale bars) and 4× (bottom 6 images with scale bars)magnification.

FIG. 21A shows images of iPSCs generated from BJ fibroblasts on feedercells. FIG. 21B shows is a larger amplification of a smaller iPSC colonyand most of its border. The iPSC colony stains positively for bothTRA-1-60 (tumor-related antigen 1-60) and OCT4. Many of the surroundingcells are also OCT4 positive. The images were taken 10 days after thelast transfection of mRNA reprogramming factors and show 10×magnification.

FIG. 22A-FIG. 22B shows an iPSC colony surrounded by fibroblasts thatexpresses Tra-1-60. FIG. 22A shows 4× magnification, FIG. 22B shows 10×magnification, and FIG. 22C shows 20× magnification.

FIG. 23A-FIG. 23J shows images of immunostained iPSCs generated from BJfibroblasts. FIG. 23A shows OCT4 staining and FIG. 23B shows TRA-1-60staining. FIG. 23C shows 20× magnification of an edge of a colone andshows high level LIN28 expression.

FIG. 23D shows LIN28 expression. It is noted that LIN28 mRNA wastransfected, but 10 days had elapsted, so this would appear to showendogenous expression. FIG. 23E shows SSEA4 expression, an importantiPSC marker. FIG. 23F shows NANOG expression and FIG. 23G shows SSEA4expression. FIG. 23H shows a second example using a small colony at 20×magnification. FIG. 23I shows NANOG expression and FIG. 23J shows SSEA4expression.

FIG. 24 shows morphological changes observed in BJ fibroblaststransitioning to iPSCs. On about Day 9, a change in morphology of BJfibroblasts was observed as the slow-growing BJ fibroblasts changed intorapidly dividing epithelial cells.

FIG. 25 shows iPSC colonies appearing on Day 16. FIG. 25A shows firstiPSC colonies appearing on Day 16 in well with no B18R protein. FIG. 25Bshows first colonies appearing on Day 16 in well with B18R protein.

FIG. 26A-FIG. 26B shows immunostaining of iPSCs one month after firstappearance of iPSC colonies. FIG. 26A shows staining for NANOG, SSEA4,and TRA-1-81, and FIG. 26B shows staining for TRA-1-60, OCT4, SSEA4, andDNMT3B.

FIG. 27A-FIG. 27J shows that iPSCs induced by RNase III-treated, cap15′-capped, 150-base poly(A)-tailed, ψ-modified mRNAs encoding a3:1:1:1:1:1 mixture of OCT4, SOX2, KLF4, LIN28, NANOG and cMYC arepluripotent based on ability to differentiate into cells of all 3 germlayers. FIG. 27A shows TUJ1 (ectoderm cells) at 4× magnification, FIG.27B shows TUJ1 at 20× magnification, FIG. 27C shows 20× magnification ofGFAP (ectoderm), FIG. 27D shows 4× magnification of NFL (ectoderm), FIG.27E shows 10× magnification of NFL, FIG. 27F shows 10× magnification ofalpha-smooth muscle actin SMA (mesoderm), FIG. 27G shows 20×magnification of Desmin muscle cells (mesoderm), FIG. 27H shows 20×magnification of SOX17 (endoderm), FIG. 27I shows 10× magnification ofAFP (endoderm), and FIG. 27J shows 10× magnification of AFP.

FIG. 28 shows alkaline phosphatase-positive colonies generated from BJfibroblasts transfected daily for 18 days with 1.2 micrograms of a3:1:1:1:1 molar ratio of HPLC-purified or RNase III-treatedpseudouridine-modified mRNAs encoding OCT4, SOX2, KLF4, LIN28 andcMYC(T58A) using the TRANSIT™ mRNA transfection reagent, with or withoutprior treatment with B18R protein. BJ fibroblasts on feeder cells weretransfected daily for 18 days, either in the presence or in the absenceof B18R protein, with 1.2 micrograms/well/day of a 3:1:1:1:1 molar ratioof ψ-modified single-stranded mRNAs encoding, respectively, OCT4, SOX2,KLF4, LIN28 and cMYC(T58) using the TransIT™ mRNA transfection reagent(Mirus Bio). In order to make the ψ-modified mRNAs substantially free ofdsRNA, the ψ-modified mRNAs were either HPLC purified or RNase IIItreated prior to being used for reprogramming. On Day 20, platescontaining iPSC colonies were fixed with 4% paraformaldehyde and stainedto detect alkaline phosphatase-positive colonies, which is indicative ofiPSC colonies. Plate A: HPLC-purified, no B18R protein; Plate B:HPLC-purified, +B18R protein; Plate C: RNase III-treated, no B18Rprotein; Plate D: RNase III-treated, +B18R protein. No alkalinephosphatase-positive colonies were present on plates of cells that weretransfected with the ψ-modified mRNAs that were not HPLC purified orRNase III treated.

FIG. 29A-FIG. 29B shows an example of a well with “too many colonies tocount.” The emerging colonies are the densely packed, rapidly dividingcells with an epithelial morphology. They no longer have the long thinBJ fibroblast morphology and the feeder cells can't be seen under theconfluent colony forming layer of cells. Basically this entire well ofcells is being reprogrammed to some extent, but not every cell willcomplete the process and form an iPSC colony. FIG. 29A shows two images,with the top image showing 4× magnification, and the bottom image blownup showing the same image with more obvious colonies outlined. FIG. 29Bshows two images, with the top image showing a single colony on abackground of fibroblast cells, and the bottom image from the edge of aparticular well which shows white rounded colonies with dark backgroundcells.

FIG. 30 shows an example of a well with efficient induction of iPSCcolonies from BJ fibroblasts transfected with pseudouridine-modifiedmRNAs encoding OCT4, SOX2, KLF4, LIN28 and cMYC(T58A), wherein the cellswere pre-treated with B18R protein prior to the transfections.

FIG. 31 shows an example of a well with efficient induction of iPSCcolonies from BJ fibroblasts transfected daily for 18 days with up to1.4 micrograms of a 3:1:1:1:1 molar ratio of unmodified mRNAs encodingOCT4, SOX2, KLF4, LIN28 and cMYC(T58A), both with and withoutpre-treatments of the cells with B18R protein prior to thetransfections. (A) 1.4 micrograms of the mRNA reprogramming mix per wellper day resulted in death of many cells, including feeder cells aroundthis iPSC colony, but some iPSC colonies survived and were propagated.(B) One microgram of unmodified mRNA reprogramming mix per well per dayresulted in less toxicity and generation of more iPS cells on Day 18.(C) Addition of B18R protein to the medium during reprogramming resultedin a confluent well of iPSC colonies—more than could be counted—and iPSCcolonies from this well maintained the morphology and growth ratesexpected for iPSCs while being propagated continuously for more than twomonths.

FIG. 32A-FIG. 32B shows images of phase contrast and both live and fixedimmunostained iPSCs generated from BJ fibroblasts using RNaseIII-treated, unmodified mRNAs encoding OCT4, SOX2, KLF4, LIN28 andcMYC(T58A) iPSC induction factors. FIG. 32A shows a phase 10×magnification, and expression of OCT4 and TRA-1-60. FIG. 32B showsexpression of NANOG, TRA-1-81, a phase 10× magnification, and expressionof SSEA4.

FIG. 33A-FIG. 33B shows images of phase contrast and fixed immunostainediPSCs generated from BJ fibroblasts using HPLC-purified, ψ-modifiedmRNAs encoding OCT4, SOX2, KLF4, LIN28 and cMYC(T58A) iPSC inductionfactors. FIG. 33A shows, 4× phase, 10 phase, 10× OCT4, and 10× TRA1-60.FIG. 33B shows 4× phase, 10× phase, 10× SOX2, 10× TRA1-80, 10× phase,and 10× NANOG.

FIG. 34 shows a qPCR gene expression assay comparison of GAPDH levelsobtained from the total cellular RNA isolated from generated iPSCcolonies with total cellular RNA isolated from BJ fibroblasts. GAPDH isa housekeeping gene, comparable in expression in both iPSC and BJfibroblast cell types. GAPDH gene expression levels were measured bytheir cycle threshold (CT) values, the PCR cycle number at which thereporter fluorescence is greater than the threshold and produces thefirst clearly detectable increase in fluorescence over background orbaseline variability. All of the traces cross the threshold (base line)at the same CT value.

FIG. 35 shows a qPCR gene expression assay comparison of CRIPTO (TDGF1),a Teratocarcinoma-derived growth factor and known pluripotency factor,obtained from cellular RNA isolated from generated iPSC colonies andcellular RNA isolated from BJ fibroblasts. CRIPTO gene expression levelswere determined by their respective CT values. The delta CT or change inexpression is 9.2 cycles, which is a 588-fold increase in expression inthe reprogramming iPSC colonies over that of the BJ fibroblasts.

FIG. 36 shows a qPCR gene expression assay comparison of NANOG, apluripotency factor involved in cell differentiation, proliferation,embryo development, somatic stem-cell maintenance, obtained fromcellular RNA isolated from generated iPSC colonies and cellular RNAisolated from BJ fibroblasts. NANOG gene expression levels weredetermined by their respective CT values. The delta CT of 7.5 cyclesrepresents a 181-fold increase in expression in the reprogrammed iPSCcolonies over that of the BJ fibroblasts.

FIG. 37 shows a qPCR gene expression assay comparison of GBX2, a DNAbinding transcription factor involved in a series of developmentalprocesses and known pluripotency factor, obtained from cellular RNAisolated from generated iPSC colonies and cellular RNA isolated from BJfibroblasts. GBX2 gene expression levels were determined by theirrespective CT values. The delta CT of 4.6 cycles represents a 24-foldincrease in expression in the reprogrammed iPSC colonies over that ofthe BJ fibroblasts.

FIG. 38 shows images of 5-factor pseudouridine-modified RNaseIII-treated KLMO3S (1:1:1:3:1) iPSCs.

FIG. 39 shows images of 5 factor pseudouridine-modified, RNaseIII-treated K₃LMO₃S (3:1:1:3:1) iPSCs.

FIG. 40 shows alkaline phosphatase-positive colonies generated from BJfibroblasts transfected with RNase III-treated mRNAs encoding KLMO₃S(1:1:1:3:1) (FIG. 40 A) and mRNAs encoding K₃LMO₃S (3:1:1:3:1) (FIG. 40B).

FIG. 41 shows alkaline phosphatase-positive colonies generated from BJfibroblasts transfected with RNase III-treated unmodified mRNAs encodingKMOS, KLMOS, and KLMNOS.

FIG. 42 shows alkaline phosphatase-positive colonies generated from BJfibroblasts transfected with RNase III-treated pseudouridine-modified,Cap0 mRNAs encoding KLMOS and KLMOS+B18R.

FIG. 43 shows alkaline phosphatase-positive colonies generated from BJfibroblasts transfected with RNase III-treated pseudouridine-modified,Cap1 mRNAs encoding KLMOS and KLMOS+B18R in FIG. 43 A; RNase III-treatedpseudouridine-modified, ARCA-capped mRNAs encoding KLMOS and KLMOS+B18Rin FIG. 43 B; and APex phosphatase treated, pseudouridine-modified,5-methylcytidine ARCA capped KLMOS and KLMOS+B18R in FIG. 43 C.

FIGS. 44A-D show alkaline phosphatase-positive colonies generated fromBJ fibroblasts transfected with mRNAs KLM_(T58A)OS (with standard1:1:1:3:1 stoichiometry) having multiple degrees of variance. FIG. 44 Ashows Wells 1-6 exhibiting the following: Well 1—mRNAs are ARCA capped;Well 2—mRNAs are ARCA capped and APex phosphatase treated; Well 3—mRNAsare ARCA capped and APex phosphatase treated+B18R protein; Well 4—mRNAsare ARCA capped and RNase III treated (2 mM Mg⁺² buffer concentration);Well 5—mRNAs are ARCA capped and RNase III treated (2 mM Mg⁺² bufferconcentration) and APex phosphatase treated; and Well 6—mRNAs are ARCAcapped and RNase III treated (2 mM Mg⁺² buffer concentration) and APexphosphatase treated+B18R protein. FIG. 44 B shows Wells 7-12 exhibitingthe following: Well 7—mRNAs are ARCA capped and RNase III treated (2 mMMg⁺² buffer concentration)+B18R protein; Well 8—mRNAs are ARCA cappedand RNase III treated (2 mM Mg⁺² buffer concentration)+B18R protein(2×); Well 9—mRNAs have a Cap0 structure; Well 10—mRNAs have a Cap0structure and RNase III treated (1 mM Mg⁺² buffer concentration); Well11—mRNAs have a Cap0 structure and RNase III treated (2 mM Mg⁺² bufferconcentration); and Well 12—mRNAs have a Cap0 structure and RNase IIItreated (1 mM Mg⁺² buffer concentration)+B18R protein. FIG. 44 C showsWells 13-18 exhibiting the following: Well 13—mRNAs have a Cap0structure and RNase III treated (2 mM Mg⁺² buffer concentration)+B18Rprotein; Well 14—mRNAs have a Cap0 structure and RNase III treated (1 mMMg⁺² buffer concentration)+B18R protein (2×); Well 15—mRNAs have a Cap0structure and RNase III treated (1 mM Mg⁺² buffer concentration)+B18Rprotein (2λ); Well 16—mRNAs have a Cap1 structure; Well 17—mRNAs have aCap1 structure and RNase III treated (1 mM Mg⁺² buffer concentration);and Well 18—mRNAs have a Cap1 structure and RNase III treated (2 mM Mg⁺²buffer concentration). FIG. 44 D shows Wells 19-24 exhibiting thefollowing: Well 19—mRNAs have a Cap1 structure and RNase III treated (1mM Mg⁺² buffer concentration)+B18R protein; Well 20—mRNAs have a Cap1structure and RNase III treated (2 mM Mg⁺² buffer concentration)+B18Rprotein; Well 21—mRNAs have a Cap1 structure and RNase III treated (1 mMMg⁺² buffer concentration)+B18R protein (2×); Well 22—mRNAs have a Cap1structure and RNase III treated (1 mM Mg⁺² buffer concentration)+B18Rprotein (2×); Well 23—mRNAs have a Cap1 structure and RNase III treated(1 mM Mg⁺² buffer concentration); and Well 24—mRNAs have a Cap1structure and RNase III treated (2 mM Mg⁺² buffer concentration).

FIGS. 45A-D show images of immunostained feeder-free reprogrammed iPScells generated from BJ fibroblasts using only Ψ-modified mRNA encodingthe five reprogramming factors, OCT4, SOX2, KLF4, LIN28, and cMYC andthen differentiated into cardiomyocytes. FIG. 45 A. shows differentiatedcells stained for class III beta-tubulin, cardiac troponinT, and sox17.FIG. 45 B shows that the iPS cells stained for pluripotency markersprior to differentiation into cardiomyocytes.

FIGS. 46A-D show images of immunostained feeder-free reprogrammed iPScells generated from BJ fibroblasts using RNase III-treated orHPLC-purified unmodified or ψ-modified mRNAs encoding iPSC inductionfactors. The differentiated cells expressed markers representing all 3germ layers of cells, including ectoderm markers, neuronal class IIIbeta-tubulin (TUJ1) (FIG. 46A), Glial Fibrillary Acidic Protein (GFAP)and neurofilament-light (NF-L) (both FIG. 46B), the mesoderm markers,alpha-smooth muscle actin (α-smooth muscle actin, α-SMA or SMA) anddesmin, and the endoderm markers, transcription factor SOX17 (FIG. 46C)and alpha fetoprotein (AFP) (shown in FIGS. 46C and D).

FIGS. 47A-B show images of immunostained feeder-free reprogrammed iPScells (11 passages) generated from BJ fibroblasts that wereHPLC-purified, mRNA III-treated mixtures that contained the shorter cMycT58A mRNA. The iPSCs stain positively for markers representing all 3germ layers of cells. Cells were found that expressed the ectodermmarker, neuronal class III beta-tubulin (TUJ1), the mesoderm markers,alpha-smooth muscle actin (SMA)(all shown in FIG. 47A) and desmin (FIG.47B), and the endoderm markers, transcription factor SOX17 (FIG. 47B)and alpha fetoprotein (AFP) (FIG. 47C).

FIGS. 48A-C show images of immunostained feeder-free reprogrammed iPScells (4 passages) generated from BJ fibroblasts that were HPLC-purifiedor RNase III-treated mRNA mixtures that contained the shorter cMyc T58AmRNA. The iPSCs stain positively for markers representing all 3 germlayers of cells. Cells were found that expressed the ectoderm markerneuronal class III beta-tubulin (TUJ1) (FIG. 48A), the mesoderm markersalpha-smooth muscle actin (SMA) (FIG. 48B) and desmin (FIG. 48C), andthe endoderm marker SOX17 (FIG. 48C).

FIGS. 49A-R show that addition of certain amounts of dsRNA inhibitsreprogramming of mouse mesenchymal stem cells to myoblasts, even though,in the absence of dsRNA, myoblasts were induced from the mesenchymalstem cells after only two daily transfections with mRNA encoding MYODprotein. This demonstrates the importance of induction of RNA sensorsand innate immune response pathways by dsRNA and the importance ofpurifying the mRNA by chromatographic, electrophoretic or other columnor gel separation methods, or treating the RNA composition or the ssRNAor mRNA composing using the RNase III treatment method disclosed herein.A) Untreated C3H10T1/2 mesenchymal stem cells (phase contrast). B)Untreated C3H10T1/2 cells (Myosin Heavy Chain, MHC in red). C) MockTransfected (phase contrast). D) Mock Transfected (MHC). E) MYOD mRNA0.5 μg/ml (phase contrast). F) MYOD mRNA 0.5 μg/ml (MHC). G) MYOD mRNA0.5 μg/ml+luc2 dsRNA 0.1 μg/ml (phase contrast). H) MYOD mRNA 0.5μg/ml+luc2 dsRNA 0.1 μg/ml (MHC). I) MYOD mRNA 0.5 μg/ml+luc2 dsRNA 0.01μg/ml (phase contrast). J) MYOD mRNA 0.5 μg/ml+luc2 dsRNA 0.01 μg/ml(MHC). K) MYOD mRNA 0.5 μg/ml+luc2 dsRNA 0.001 μg/ml (phase contrast).L) MYOD mRNA 0.5 μg/ml+luc2 dsRNA 0.00001 μg/ml (MHC). M) MYOD mRNA 0.5μg/ml+luc2 dsRNA 0.0001 μg/ml (phase contrast). N) MYOD mRNA 0.5μg/ml+luc2 dsRNA 0.0001 μg/ml (MHC). O) MYOD mRNA 0.5 μg/ml+luc2 dsRNA0.00001 μg/ml (phase contrast). P) MYOD mRNA 0.5 μg/ml+luc2 dsRNA0.00001 μg/ml (MHC). Q) MYOD mRNA 0.5 μg/ml+luc2 dsRNA 0.000001 μg/ml(phase contrast). R) MYOD mRNA 0.5 μg/ml+luc2 dsRNA 0.000001 μg/ml(MHC).

FIGS. 50A-B show 10× phase contrast images of fibroblast cells that weretransfected with either ψ-mRNAs encoding only A and N proteins (top) orψ-mRNAs encoding AMNP proteins (bottom). FIG. 50A shows 10× phase IMR90Mock transfected fibroblasts with original cell morphology. FIG. 50Bshows 10× phase of cells transfected with RNase II-treated ψ-modifiedmRNAs encoding AMNP with neuron morphology.

FIGS. 51A-B show a 20× phase contrast image of the morphology exhibitedby the reprogrammed fibroblast cells on day 7 (top; 51A). In this case,the top image shows the fibroblast cells that were transfected withψ-mRNAs encoding AMNP proteins and the bottom image (51B) shows thefibroblast cells that were transfected with ψ-mRNAs encoding only A andN proteins. After fixation, the cells in the top image stainedpositively for microtubule-associated protein-2 (MAP2), a pan-neuronalmarker.

DEFINITIONS

The present invention will be understood and interpreted based on termsas defined below.

The terms “comprising”, “containing”, “having”, “include”, and“including” are to be construed as “including, but not limited to”unless otherwise noted. The terms “a,” “an,” and “the” and similarreferents in the context of describing the invention and, specifically,in the context of the appended claims, are to be construed to cover boththe singular and the plural unless otherwise noted. The use of any andall examples or exemplary language (“for example”, “e.g.”, “such as”) isintended merely to illustrate aspects or embodiments of the invention,and is not to be construed as limiting the scope thereof, unlessotherwise claimed.

When the terms “about” or “approximately” are used herein to describe anumber or quantity, the term shall be interpreted to mean the specifiednumber or quantity plus or minus 20% of that number or quantity. Forexample, the statements “about 1 mM to 4 mM” or “about 1 to 4 mM” shallbe interpreted to mean from 0.8 mM to 4.8 mM.”

With respect to the use of the word “derived”, such as for an RNA(including ssRNA or mRNA) or a polypeptide that is “derived” from asample, biological sample, cell, tumor, or the like, it is meant thatthe RNA or polypeptide either was present in the sample, biologicalsample, cell, tumor, or the like, or was made using the RNA in thesample, biological sample, cell, tumor, or the like by a process such asan in vitro transcription reaction, or an RNA amplification reaction,wherein the RNA or polypeptide is either encoded by or a copy of all ora portion of the RNA or polypeptide molecules in the original sample,biological sample, cell, tumor, or the like. By way of example, such RNAcan be from an in vitro transcription or an RNA amplification reaction,with or without cloning of cDNA, rather than being obtained directlyfrom the sample, biological sample, cell, tumor, or the like, so long asthe original RNA used for the in vitro transcription or an RNAamplification reaction was from the sample, biological sample, cell,tumor, or the like. In most embodiments of the present invention, assRNA or mRNA that is derived from a biological sample, cell, tumor, orthe like is amplified from mRNA in the biological sample, cell, tumor,or the like using an RNA amplification reaction comprising in vitrotranscription, as described elsewhere herein.

With respect embodiments of the present invention pertaining to themethods, compositions, systems and kits for introducing an RNAcomposition comprising in vitro-synthesized ssRNA or mRNA encoding oneor more proteins into a human or animal (e.g., mammalian) cell (e.g., acell that is ex vivo in culture or in vivo in a tissue, organ ororganism) to induce a biological or biochemical effect, the terms“biological or biochemical effect” or “biological effect” or“biochemical effect” herein mean and refer to any effect in the cellinto which the RNA composition is introduced or any effect in a tissue,organ or organism containing the cell into which the RNA composition isintroduced, which effect would be expected or anticipated or understoodby a person with knowledge in the art based on information and knowledgein the art about the protein encoded by said ssRNA or mRNA. For example,in some embodiments wherein the RNA comprises ssRNA or mRNA that encodesa wild-type non-mutated protein that has a known function (e.g., as anenzyme, growth factor, a cell surface receptor e.g., in a cell signalingpathway, a cytokine, a chemokine, or as an effector molecule in anactive or innate immune response mechanism), the biological orbiochemical effect of said introducing of said RNA composition into acell that has a defective or non-functional mutant gene, wherein thecell's own protein is defective or non-functional in said cell, would bethat the introduced RNA composition may substitute for or replace orcomplement the cell's defective or non-functional protein, therebyrestoring the normal biological or biochemical effect of the wild-typeprotein encoded by the RNA composition comprising ssRNA or mRNA. By wayof further example, in some embodiments wherein an mRNA encodingerythropoietin is introduced into a mammal cell in vivo in a mammal, onebiological or biochemical effect is an increase in the hematocrit orerythrocyte volume fraction (EVF), reflecting an increase in the volumepercentage (%) of red blood cells in blood of said mammal. Thus,although the present invention provides a method for inducing a broadrange of biological or biochemical effects, those biological orbiochemical effects are predictable and will be understood by those withknowledge in the art based on reading the description of the presentinventions, and therefore are within the scope and coverage of thepresent invention.

The terms “sample” and “biological sample” are used in their broadestsense and encompass samples or specimens obtained from any source thatcontains or may contain eukaryotic cells, including biological andenvironmental sources. As used herein, the term “sample” when used torefer to biological samples obtained from organisms, includes bodilyfluids (e.g., blood or saliva), feces, biopsies, swabs (e.g., buccalswabs), isolated cells, exudates, and the like. The organisms includeanimals and humans. However, these examples are not to be construed aslimiting the types of samples or organisms that find use with thepresent invention. In addition, in order to perform research or studythe results related to use of a method or composition of the invention,in some embodiments, a “sample” or “biological sample” comprises fixedcells, treated cells, cell lysates, and the like. In some embodiments,such as embodiments of the method wherein the mRNA is delivered into acell from an organism that has a known disease or into a cell thatexhibits a disease state or a known pathology, the “sample” or“biological sample” also comprises bacteria or viruses.

As used herein, the term “incubating” and variants thereof meancontacting one or more components of a reaction with another componentor components, under conditions and for sufficient time such that adesired reaction product is formed.

“In vitro” herein refers to an artificial environment and to processesor reactions that occur within an artificial environment. In vitroenvironments can be composed of, but are not limited to, processes orreactions that occur in a test tube. The term “in vivo” refers to thenatural environment and to processes or reactions that occur within anatural environment (e.g., in a living cell or a human or animal). “Exvivo” herein refers to processes or reactions that occur within a cellin culture.

As used herein, a “nucleoside” refers to a molecule composed of anucleic acid base (e.g., the canonical nucleic acid bases: guanine (G),adenine (A), thymine (T), uracil (U), or cytidine (C), or a modifiednucleic acid base (e.g., 5-methylcytosine (m⁵C)), that is covalentlylinked to a pentose sugar (e.g., ribose or 2′-deoxyribose). A nucleosidecan also be modified. For example, pseudouridine (abbreviated by theGreek letter psi or Ψ) is a modified nucleoside composed of ribose whichis linked to a carbon of uracil, whereas the canonical nucleosideuridine is linked to a nitrogen designated as the 1 position of uracil.A “nucleotide” or “mononucleotide” refers to a nucleoside that isphosphorylated at one or more of the hydroxyl groups of the pentosesugar. The number of phosphate groups can also be indicated (e.g., a“mononucleotide” is composed of a nucleoside that is phosphorylated atone of the hydroxyl groups of the pentose sugar).

Linear nucleic acid molecules are said to have a “5′ terminus” (5′ end)and a “3′ terminus” (3′ end) because, during synthesis (e.g., by a DNAor RNA polymerase (the latter process being referred to as“transcription”), mononucleotides are joined in one direction via aphosphodiester linkage to make oligonucleotides or polynucleotides, in amanner such that a phosphate on the 5′ carbon of one mononucleotidesugar moiety is joined to an oxygen on the 3′ carbon of the sugar moietyof its neighboring mononucleotide. Therefore, an end of a linearsingle-stranded oligonucleotide or polynucleotide or an end of onestrand of a linear double-stranded nucleic acid (RNA or DNA) is referredto as the “5′ end” if its 5′ phosphate is not joined or linked to theoxygen of the 3′ carbon of a mononucleotide sugar moiety, and as the “3′end” if its 3′ oxygen is not joined to a 5′ phosphate that is joined toa sugar moiety of a subsequent mononucleotide. A terminal nucleotide, asused herein, is the nucleotide at the end position of the 3′ or 5′terminus.

In order to accomplish specific goals, a nucleic acid base, sugarmoiety, or internucleoside (or internucleotide) linkage in one or moreof the nucleotides of the mRNA that is introduced into a eukaryotic cellin the methods of the invention may comprise a modified base, sugarmoiety, or internucleoside linkage. For example, in addition to theother modified nucleotides discussed elsewhere herein for performing themethods of the present invention, one or more of the nucleotides of themRNA can also have a modified nucleic acid base comprising or consistingof: xanthine; allyamino-uracil; allyamino-thymidine; hypoxanthine;2-aminoadenine; 5-propynyl uracil; 5-propynyl cytosine; 4-thiouracil;6-thioguanine; an aza or deaza uracil; an aza or deaza thymidine; an azaor deaza cytosines; an aza or deaza adenine; or an aza or deazaguanines; or a nucleic acid base that is derivatized with a biotinmoiety, a digoxigenin moiety, a fluorescent or chemiluminescent moiety,a quenching moiety or some other moiety in order to accomplish one ormore specific other purposes; and/or one or more of the nucleotides ofthe mRNA can have a sugar moiety, such as, but not limited to:2′-fluoro-2′-deoxyribose or 2′-O-methyl-ribose, which provide resistanceto some nucleases; or 2′-amino-2′-deoxyribose or2′-azido-2′-deoxyribose, which can be labeled by reacting them withvisible, fluorescent, infrared fluorescent or other detectable dyes orchemicals having an electrophilic, photoreactive, alkynyl, or otherreactive chemical moiety.

In some embodiments of the methods, compositions or kits of theinvention, one or more of the nucleotides of the mRNA comprises amodified internucleoside linkage, such as a phosphorothioate,phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkage,which are resistant to some nucleases, including in a thio-ARCAdinucleotide cap analog (Grudzien-Nogalska et al. 2007) that is used inan IVT reaction for co-transcriptional capping of the RNA, or in thepoly(A) tail (e.g., by incorporation of a nucleotide that has themodified phosphorothioate, phosphorodithioate, phosphoroselenate, orphosphorodiselenate linkage during IVT of the RNA or, e.g., byincorporation of ATP that contains the modified phosphorothioate,phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkageinto a poly(A) tail on the RNA by polyadenylation using a poly(A)polymerase). The invention is not limited to the modified nucleic acidbases, sugar moieties, or internucleoside linkages listed, which arepresented to show examples which may be used for a particular purpose ina method.

As used herein, a “nucleic acid” or a “polynucleotide” or an“oligonucleotide” is a polymer molecule comprising a covalently linkedsequence or series of “mononucleosides,” also referred to as“nucleosides,” in which the 3′-position of the pentose sugar of onenucleoside is linked by an internucleoside linkage, such as, but notlimited to, a phosphodiester bond, to the 5′-position of the pentosesugar of the next nucleoside (i.e., a 3′ to 5′ phosphodiester bond), andin which the nucleotides are linked in specific sequence; i.e., a linearorder of nucleotides. In some embodiments, the oligonucleotide consistsof or comprises ribonucleotides (“RNA”). A nucleoside linked to aphosphate group is referred to as a “nucleotide.” The nucleotide that islinked to the 5′-position of the next nucleotide in the series isreferred to as “5′ of” or the “5′ nucleotide” and the nucleotide that islinked to the 3′-position of the 5′ nucleotide is referred to as “3′ of”or the “3′ nucleotide.” The terms “3′-of” and “5′-of” are used hereinwith respect to the present invention to refer to the position ororientation of a particular nucleic acid sequence or genetic elementwithin a strand of the particular nucleic acid, polynucleotide, oroligonucleotide being discussed (such as an RNA polymerase promoter,start codon, open reading frame, or stop codon relative to othersequences or genetic elements within a DNA strand; or a cap nucleotide,5′ or 3′ untranslated region (5′ UTR or 3′ UTR), Kozak sequence, startcodon, coding sequence, stop codon, or poly-A tail relative to othersequences within an mRNA strand). Thus, although the synthesis of RNA ina 5′-to-3′ direction during transcription is thought of as proceeding ina “downstream” direction, the sense promoter sequence exhibited by anRNA polymerase promoter is referred to herein as being 3-of thetranscribed template sequence on the template strand. Those withknowledge in the art will understand these terms in the context ofnucleic acid chemistry and structure, particularly related to the 3′-and 5′-positions of sugar moieties of canonical nucleic acidnucleotides. By way of further example, a first sequence that is “5′-of”a second sequence means that the first sequence is exhibited at orcloser to the 5′-terminus relative to the second sequence. If a firstnucleic acid sequence is 3′-of a second sequence on one strand, thecomplement of the first sequence will be 5′-of the complement of thesecond sequence on the complementary strand.

Also, for a variety of reasons, a nucleic acid or polynucleotide of theinvention may comprise one or more modified nucleic acid bases, sugarmoieties, or internucleoside linkages. By way of example, some reasonsfor using nucleic acids or polynucleotides that contain modified bases,sugar moieties, or internucleoside linkages include, but are not limitedto: (1) modification of the T_(m); (2) changing the susceptibility ofthe polynucleotide to one or more nucleases; (3) providing a moiety forattachment of a label; (4) providing a label or a quencher for a label;or (5) providing a moiety, such as biotin, for attaching to anothermolecule which is in solution or bound to a surface. For example, insome embodiments, an oligonucleotide, such as the terminal taggingoligoribonucleotide, may be synthesized so that the random 3′-portioncontains one or more conformationally restricted ribonucleic acidanalogs, such as, but not limited to one or more ribonucleic acidanalogs in which the ribose ring is “locked” with a methylene bridgeconnecting the 2′-O atom with the 4′-C atom (e.g., as available fromExiqon, Inc. under the trademark of “LNA™”); these modified nucleotidesresult in an increase in the T_(m) or melting temperature by about 2degrees to about 8 degrees centigrade per nucleotide monomer. If theT_(m) is increased, it might be possible to reduce the number of randomnucleotides in the random 3′-portion of the terminal taggingoligoribonucleotide. However, a modified nucleotide, such as an LNA mustbe validated to function in the method for its intended purpose, as wellas satisfying other criteria of the method; for example, in someembodiments, one criterium for using the modified nucleotide in themethod is that the oligonucleotide that contains it can be digested by asingle-strand-specific RNase.

In order to accomplish the goals of the invention, by way of example,but not of limitation, the nucleic acid bases in the mononucleotides maycomprise guanine, adenine, uracil, thymine, or cytidine, oralternatively, one or more of the nucleic acid bases may comprise amodified base, such as, but not limited to xanthine, allyamino-uracil,allyamino-thymidine, hypoxanthine, 2-aminoadenine, 5-propynyl uracil,5-propynyl cytosine, 4-thiouracil, 6-thioguanine, aza and deaza uracils,thymidines, cytosines, adenines, or guanines. Still further, they maycomprise a nucleic acid base that is derivatized with a biotin moiety, adigoxigenin moiety, a fluorescent or chemiluminescent moiety, aquenching moiety or some other moiety. The invention is not limited tothe nucleic acid bases listed; this list is given to show an example ofthe broad range of bases which may be used for a particular purpose in amethod.

With respect to nucleic acids or polynucleotides of the invention, oneor more of the sugar moieties can comprise ribose or 2′-deoxyribose, oralternatively, one or more of the sugar moieties can be some other sugarmoiety, such as, but not limited to, 2′-fluoro-2′-deoxyribose or2′-O-methyl-ribose, which provide resistance to some nucleases, or2′-amino-2′-deoxyribose or 2′-azido-2′-deoxyribose, which can be labeledby reacting them with visible, fluorescent, infrared fluorescent orother detectable dyes or chemicals having an electrophilic,photoreactive, alkynyl, or other reactive chemical moiety.

The internucleoside linkages of nucleic acids or polynucleotides of theinvention can be phosphodiester linkages, or alternatively, one or moreof the internucleoside linkages can comprise modified linkages, such as,but not limited to, phosphorothioate, phosphorodithioate,phosphoroselenate, or phosphorodiselenate linkages, which are resistantto some nucleases

Oligonucleotides and polynucleotides, including chimeric (i.e.,composite) molecules and oligonucleotides with modified bases, sugars,or internucleoside linkages are commercially available (e.g., TriLinkBiotechnologies, San Diego, Calif., USA or Integrated DNA Technologies,Coralville, Iowa).

Whenever we refer to an “RNase III-treated” sample or composition (e.g.,an “RNase III-treated” RNA composition, ssRNA, capped and/orpolyadenylated ssRNA, mRNA, ssRNA or mRNA, in vitro-transcribed ssRNA,IVT RNA, or the like), we mean that the sample or other composition thatcontains or may contain dsRNA has been treated with RNase III using anRNase III treatment or an “RNase III treatment method.”

Whenever we refer to an “RNase III treatment” or “RNase III treatmentmethod” or “treating a sample or composition with RNase III” herein, wemean incubating a sample or composition comprising ssRNA and whichcontains or may contain dsRNA (e.g., an RNA composition, ssRNA, cappedand/or polyadenylated ssRNA, mRNA, ssRNA or mRNA, in vitro-transcribedssRNA, IVT RNA, or the like) with RNase III enzyme in a buffered aqueoussolution or reaction mixture under conditions wherein the RNase III isactive [e.g., wherein the buffered aqueous solution has a pH of about pH7 to pH 9 and comprises a salt or other compound at sufficientconcentration to maintain an ionic strength equivalent to at least 50 mMpotassium acetate or potassium glutamate (e.g., about 50-300 mMpotassium acetate or potassium glutamate), and a magnesium compound thatprovides about 1 mM to about 4 mM of initially non-chelated divalentmagnesium cations] and then optionally, in some embodiments, cleaning upthe ssRNA in the sample or composition to remove the RNase III enzymeand/or nucleotides and/or small oligonucleotides and/or salt, and/orother RNase III treatment reaction components. In some embodiments ofthe RNase III treatment or RNase III treatment method or treating of asample or composition with RNase III, the RNA quick cleanup methoddescribed herein is used for said cleaning up of the ssRNA in the sampleor composition. However, in other embodiments another cleanup method isused for said cleaning up of the ssRNA.

The terms “purified” or “to purify” or “cleaned up” or “to clean up”herein refers to the removal of components (e.g., contaminants) from asample (e.g., from in vitro-transcribed or in vitro-synthesized ssRNA,mRNA or a precursor thereof). For example, nucleic acids, such as invitro-transcribed or in vitro-synthesized ssRNA, mRNA or a precursorthereof) are purified or cleaned up by removal of contaminating proteinsin the in vitro transcription reaction mixture, or undesired nucleicacid species (e.g., the DNA template, or RNA contaminants other than thedesired ssRNA or mRNA, such as dsRNA, or in vitro transcription productswhich are shorter or longer than the desired full-length ssRNA or mRNAencoded by the template. The removal of contaminants results in anincrease in the percentage of desired nucleic acid (e.g., the desiredssRNA or mRNA) comprising the nucleic acid. The terms “purified” or “topurify,” when used herein, refer to use of methods to removecontaminants by use of a chromatographic or electrophoretic separationdevice comprising a resin, matrix or gel or the like (e.g., by HPLC,FPLC or gravity flow column chromatography, or agarose or polyacrylamideelectrophoresis”). In contrast, the terms “cleaned up” and “to cleanup,” when used herein, refer to use of methods to remove contaminants byextraction (e.g., organic solvent extraction, e.g., phenol and/orchloroform extraction), precipitation (e.g., precipitation of RNA withammonium acetate), and washing of precipitates (e.g., washing of RNAprecipitates with 70% ethanol), without use of a chromatographic orelectrophoretic separation device comprising a resin, matrix or gel orthe like. Thus, when a sample (e.g., in vitro-transcribed or invitro-synthesized ssRNA or mRNA) is cleaned up, said method, in certainembodiments, is much easier, faster, much less expensive, required muchless knowledge and training, and requires fewer and less expensivematerials and less labor than would be required to purify the sample. Insome other embodiments, a sample (e.g., in vitro-transcribed or invitro-synthesized ssRNA or mRNA or a precursor thereof) is furthercleaned up or purified using a rapid gel filtration method with across-linked dextran (e.g., Sephadex, e.g., a Sephadex spin column) inorder to separate low molecular weight molecules, such as salts,buffers, nucleotides and small oligonucleotides, solvents (e.g., phenol,chloroform) or detergents from the ssRNA or mRNA.

The invention is not limited with respect to an RNA polymerase used forin vitro transcription or synthesis of a ssRNA or mRNA used in a methodor comprising a composition, system or kit of the present invention.However, in some preferred embodiments, the ssRNA or mRNA is synthesizedusing a T7-type RNA polymerase. A “17-type RNA polymerase” (or “T7RNAP”) herein means 17 RNA polymerase (e.g., see Studier, F W et al.,pp. 60-89 in Methods in Enzymology, Vol. 185, ed. by Goeddel, DV,Academic Press, 1990) or an RNAP derived from a “T7-type” bacteriophage,meaning a bacteriophage that has a similar genetic organization to thatof bacteriophage T7. The genetic organization of all T7-type phages thathave been examined has been found to be essentially the same as that ofT7. Examples of T7-type bacteriophages according to the inventioninclude Escherichia coli phages such as T3 and Salmonella typhimuriumphages such as SP6, and Klebsiella phages such as K11 (McAllister W Tand Raskin C A, 1993), as well as mutant forms of such RNAPs (e.g.,Sousa et al., U.S. Pat. No. 5,849,546; Padilla, R and Sousa, R, NucleicAcids Res., 15: e138, 2002; Sousa, R and Mukherjee, S, Prog Nucleic AcidRes Mol Biol., 73: 1-41, 2003; Guillerez, J, et al., U.S. Pat. No.7,335,471 or U.S. Patent Application No. 20040091854). Thus, in somepreferred embodiments of the methods wherein an RNA polymerase is usedfor in vitro transcription or synthesis of any ssRNA used in a method orcomposition herein, the RNA polymerase is selected from the groupconsisting of T7 RNAP, T3 RNAP, SP6 RNAP wild-type 17-type RNAPs, theY639F mutant of T7 RNAP, the Y640F mutant of T3 RNAP, the Y631F mutantof SP6 RNAP, the Y662F mutant of Klebsiella phage K11 RNAP, theY639F/H784A double-mutant of T7 RNAP, the P266L mutant of T7 RNAP, theP267L mutant of T3 RNAP, and the P239L mutant of SP6 RNAP, and the P289Lmutant of Klebsiella phage K11 RNAP. However, in other embodiments, thessRNA or mRNA is synthesized using another RNA polymerase that binds andinitiates transcription at an RNA polymerase promoter that is joined toa coding sequence in the DNA template which that results in synthesis ofthe ssRNA or mRNA by said RNA polymerase.

A “template” is a nucleic acid molecule that serves to specify thesequence of nucleotides exhibited by a nucleic that is synthesized by aDNA-dependent or RNA-dependent nucleic acid polymerase. If the nucleicacid comprises two strands (i.e., is “double-stranded”), and sometimeseven if the nucleic acid comprises only one strand (i.e., is“single-stranded”), the strand that serves to specify the sequence ofnucleotides exhibited by a nucleic that is synthesized is the “template”or “the template strand.” The nucleic acid synthesized by the nucleicacid polymerase is complementary to the template. Both RNA and DNA arealways synthesized in the 5′-to-3′ direction, beginning at the 3′-end ofthe template strand, and the two strands of a nucleic acid duplex alwaysare aligned so that the 5′ ends of the two strands are at opposite endsof the duplex (and, by necessity, so then are the 3′ ends). A primer isrequired for both RNA and DNA templates to initiate synthesis by a DNApolymerase, but a primer is not required to initiate synthesis by aDNA-dependent RNA polymerase, which is usually called simply an “RNApolymerase.”

“Transcription” or “in vitro transcription” or “IVT” means the formationor synthesis of an RNA molecule by an RNA polymerase using a DNAmolecule as a template using an in vitro reaction or process.

An “RNA polymerase promoter” or a “promoter,” as used herein, means asegment of DNA that exhibits a nucleotide sequence to which an RNApolymerase that recognizes said sequence is capable of binding andinitiating synthesis of RNA. The RNA polymerase that recognizes thepromoter may also be designated (e.g., a “T7 promoter” or a “T7 RNApolymerase promoter” or a “T7 RNAP promoter” is a promoter recognized byT7 RNA polymerase). Most, but not all, RNA polymerase promoters aredouble-stranded. If an RNA polymerase promoter is double-stranded, theRNA polymerase promoter exhibits (or has) a “sense promoter sequence”and an “anti-sense promoter sequence.” As used herein, the “sensepromoter sequence” is defined as the sequence of an RNA polymerasepromoter that is joined to the template strand, in which case the sensepromoter sequence is 3′-of the DNA sequence in the template strand thatserves to specify the sequence of nucleotides exhibited by the RNA thatis synthesized by the RNA polymerase that recognizes and binds to theRNA polymerase promoter. As used herein, the “anti-sense promotersequence” is defined as the sequence of an RNA polymerase promoter thatis complementary to the sense promoter sequence. If an RNA polymerase(e.g., phage N4 RNA polymerase) can synthesize RNA using asingle-stranded RNA polymerase promoter, then the RNA polymerasepromoter exhibits only the sense promoter sequence. It should be notedthat the definitions of a “sense promoter sequence” and “anti-sensepromoter sequence” may be the opposite of what would be expected by somepeople with knowledge in the art, but the terminology used herein wasdeveloped in the relatively new context of single-stranded RNApolymerase promoters. It is more easily understood and remembered bynoting that a sense promoter sequence in the template strand (i.e.,joined to the 3′-termini of the first-strand cDNA molecules) results insynthesis of sense RNA using the methods of the invention.

A “cap” or a “cap nucleotide” means a nucleoside-5′-triphosphate that,under suitable reaction conditions, is used as a substrate by a cappingenzyme system and that is thereby joined to the 5′-end of an uncappedRNA comprising primary RNA transcripts (which have a 5′-triphosphate) orRNA having a 5′-diphosphate. The nucleotide that is so joined to the RNAis also referred to as a “cap nucleotide” herein. A “cap nucleotide” isa guanine nucleotide that is joined through its 5′ end to the 5′ end ofa primary RNA transcript. The RNA that has the cap nucleotide joined toits 5′ end is referred to as “capped RNA” or “capped RNA transcript” or“capped transcript.” A common cap nucleoside is 7-methylguanosine orN⁷-methylguanosine (sometimes referred to as “standard cap”), which hasa structure designated as “m⁷G,” in which case the capped RNA or“m⁷G-capped RNA” has a structure designated asm⁷G(5′)ppp(5′)N₁(pN)_(x)-OH(3′), or more simply, as m⁷GpppN₁(pN)_(x) orm⁷G[5′]ppp[5′]N, wherein m⁷G represents the 7-methylguanosine capnucleoside, ppp represents the triphosphate bridge between the 5′carbons of the cap nucleoside and the first nucleotide of the primaryRNA transcript, N₁(pN)_(x)-OH(3′) represents the primary RNA transcript,of which N₁ is the most 5′-nucleotide, “p” represents a phosphate group,“G” represents a guanosine nucleoside, “m” represents the methyl groupon the 7-position of guanine, and “[5′]” indicates the position at whichthe “p” is joined to the ribose of the cap nucleotide and the firstnucleoside of the mRNA transcript (“N”). In addition to this “standardcap,” a variety of other naturally-occurring and synthetic cap analogsare known in the art. RNA that has any cap nucleotide is referred to as“capped RNA.”

A capped RNA comprising a composition or system or kit or used in amethod of the present invention is synthesized in vitro. In someembodiments, the capped RNA is synthesized post-transcriptionally fromin vitro-transcribed RNA, by capping ssRNA that has a 5′ triphosphategroup or ssRNA that has a 5′ diphosphate group using a capping enzymesystem (e.g., using a capping enzyme system comprising an RNAguanyltransferase; e.g., vaccinia capping enzyme system or Saccharomycescerevisiae capping enzyme system).

Alternatively, in some other embodiments, the capped RNA is synthesizedco-transcriptionally by in vitro transcription (IVT) of a DNA templatethat contains an RNA polymerase promoter, wherein, in addition to theGTP, the IVT reaction also contains a dinucleotide cap analog (e.g., am⁷GpppG cap analog or an N⁷-methyl, 2′-O-methyl-GpppG ARCA cap analog oran N⁷-methyl, 3′-O-methyl-GpppG ARCA cap analog) or a phosphorothioatedinucleotide cap analog or thio-ARCA (Grudzien-Nogalska E, et al., 2007)using methods known in the art (e.g., using an AMPLICAP™ T7 capping kitfor making an m⁷GpppG-capped RNA, or, e.g., using an INCOGNITO™ T7 ARCA5mC- & Ψ-RNA transcription kit or a MESSAGEMAX™ T7 ARCA-capped messagetranscription kit for making an ARCA-capped RNA, CELLSCRIPT, INC,Madison, Wis., USA). However, some embodiments of methods, compositionsor systems or kits (e.g, in methods wherein the mRNA used for saidintroducing of mRNA encoding at least one reprogramming factor into acell that exhibits a first differentiated state or phenotype, whereinthe ssRNA or mRNA was capped co-transcriptionally using a cap analog),the ssRNA or mRNA is further treated with a phosphatase (e.g., asdescribed elsewhere herein) to remove RNA molecules that exhibit a5′-triphosphate group.

Post-transcriptional capping of a 5′-triphosphorylated primary mRNAtranscript in vivo (or using a capping enzyme system in vitro) occursvia several enzymatic steps (Higman et al., 1992, Martin et al., 1975,Myette and Niles, 1996).

The following enzymatic reactions are generally involved in capping ofeukaryotic mRNA:

(1) RNA triphosphatase cleaves the 5′-triphosphate of mRNA to adiphosphate,

pppN₁(p)N_(x)-OH(3′)→ppN₁(pN)_(x)-OH(3′)+Pi; and then

(2) RNA guanyltransferase catalyzes joining of GTP to the 5′-diphosphateof the most 5′ nucleotide (N₁) of the mRNA,

ppN₁(pN)_(x)-OH(3′)+GTP→G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+PPi; and finally,

(3) guanine-7-methyltransferase, using S-adenosyl-methionine (AdoMet) asa co-factor, catalyzes methylation of the 7-nitrogen of guanine in thecap nucleotide,

G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+AdoMet→m⁷G(5′)ppp(5′)N₁(pN)_(x)-OH(3′)+AdoHyc.

RNA that results from the action of the RNA triphosphatase and the RNAguanyltransferase enzymatic activities, as well as RNA that isadditionally methylated by the guanine-7-methyltransferase enzymaticactivity, is referred to herein as “5′ capped RNA” or “capped RNA”, anda “capping enzyme system comprising RNA guanyltransferase” or, moresimply, a “capping enzyme system” or a “capping enzyme” herein means anycombination of one or more polypeptides having the enzymatic activitiesthat result in “capped RNA.” Capping enzyme systems, including clonedforms of such enzymes, have been identified and purified from manysources and are well known in the art (Banerjee 1980, Higman et al.,1992 and 1994; Myette and Niles 1996, Shuman 1995 and 2001; Shuman etal. 1980; Wang et al. 1997). Any capping enzyme system that can convertuncapped RNA that has a 5′ polyphosphate to capped RNA can be used toprovide a capped RNA for any of the embodiments of the presentinvention. In some embodiments, the capping enzyme system is a poxviruscapping enzyme system. In some preferred embodiments, the capping enzymesystem is vaccinia virus capping enzyme. In some embodiments, thecapping enzyme system is Saccharomyces cerevisiae capping enzyme. Also,in view of the fact that genes encoding RNA triphosphatase, RNAguanyltransferase and guanine-7-methyltransferase from one source cancomplement deletions in one or all of these genes from another source,the capping enzyme system can originate from one source, or one or moreof the RNA triphosphatase, RNA guanyltransferase, and/orguanine-7-methyltransferase activities can comprise a polypeptide from adifferent source.

The RNA compositions comprising ssRNA or mRNA used in the methods of thepresent invention can exhibit a modified cap nucleotide; in someembodiments, the ssRNA molecules are capped using a capping enzymesystem as described by Jendrisak; J et al. in U.S. patent applicationSer. No. 11/787,352 (Publication No. 20070281336, herein incorporated byreference). A “modified cap nucleotide” of the present invention means acap nucleotide wherein the sugar, the nucleic acid base, or theinternucleoside linkage is chemically modified compared to thecorresponding canonical 7-methylguanosine cap nucleotide. Examples of amodified cap nucleotide include a cap nucleotide comprising: (i) amodified 2′- or 3′-deoxyguanosine-5′-triphosphate (or guanine 2′- or3′-deoxyribonucleic acid-5′-triphosphate) wherein the 2′- or 3′-deoxyposition of the deoxyribose sugar moiety is substituted with a groupcomprising an amino group, an azido group, a fluorine group, a methoxygroup, a thiol (or mercapto) group or a methylthio (or methylmercapto)group; or (ii) a modified guanosine-5′-triphosphate, wherein the 06oxygen of the guanine base is substituted with a methyl group; or (iii)3′-deoxyguanosine. For the sake of clarity, it will be understood hereinthat an “alkoxy-substituted deoxyguanosine-5′-triphosphate” can also bereferred to as an “O-alkyl-substituted guanosine-5′-triphosphate”; byway of example, but without limitation,2′-methoxy-2′-deoxyguanosine-5′-triphosphate (2′-methoxy-2′-dGTP) and3′-methoxy-3′-deoxyguanosine-5′-triphosphate (3′-methoxy-3′-dGTP) canalso be referred to herein as 2′-O-methylguanosine-5′-triphosphate(2′-OMe-GTP) and 3′-O-methylguanosine-5′-triphosphate (3′-OMe-GTP),respectively. Following joining of the modified cap nucleotide to the5′-end of the uncapped RNA comprising primary RNA transcripts (or RNAhaving a 5′-diphosphate), the portion of said modified cap nucleotidethat is joined to the uncapped RNA comprising primary RNA transcripts(or RNA having a 5′-diphosphate) may be referred to herein as a“modified cap nucleoside” (i.e., without referring to the phosphategroups to which it is joined), but sometimes it is referred to as a“modified cap nucleotide”.

A “modified-nucleotide-capped RNA” is a capped RNA molecule that issynthesized using a capping enzyme system and a modified cap nucleotide,wherein the cap nucleotide on its 5′ terminus comprises the modified capnucleotide, or a capped RNA that is synthesize co-transcriptionally inan in vitro transcription reaction that contains a modified dinucleotidecap analog wherein the dinucleotide cap analog contains the chemicalmodification in the cap nucleotide. In some embodiments, the modifieddinucleotide cap analog is an anti-reverse cap analog or ARCA or athio-ARCA (Grudzien et al. 2004, Grudzien-Nogalska et al., 2007,Jemielity et al. 2003, Peng et al. 2002, Stepinski et al. 2001).

A “primary RNA” or “primary RNA transcript” means an RNA molecule thatis synthesized by an RNA polymerase in vivo or in vitro and which RNAmolecule has a triphosphate on the 5′-carbon of its most 5′ nucleotide.

Human and animal cells possess wide array of defense mechanismscomprising RNA sensors and signaling pathways to protect them againstexogenous introduction of RNA. It is important to understand thesecellular defense mechanisms and take them into account when designingRNA molecules to be introduced into a human or animal cell to reprogramthe cell to another state of differentiation or phenotype (e.g., forclinical research or for regenerative medicine or immunotherapy) so thatthose RNA molecules avoid or minimize induction and/or activation of thenumerous RNA sensors and signaling pathways. Among these are “dsRNA RNAsensors” and “dsRNA signaling pathways,” which means and includes any ofthe mechanisms by which a human or animal cell recognizes and respondsto dsRNA that is introduced into the cell, such as dsRNA that isintroduced into the cell as a result of infection by virus. Inparticular, induction of the interferon (IFN) system by dsRNA is theprime activator of a mammalian cell's response to detection of dsRNA bycellular RNA sensors (e.g., see Gantier, M P and Williams, B R G, 2007,and Jiang, F et al. 2011, both incorporated herein by reference in theirentirety). Following its activation by dsRNA, type-I IFN induces andactivates a Ser/Thr protein kinase now commonly known as PKR (formerlyalso known as Eiflak2, Prkr, Tik, DAI, P1-eIF-2, and p68 kinase). PKRinhibits mRNA translation by catalyzing phosphorylation of the alphasubunit of the eukaryotic translation initiation factor 2 (eIF-2a).Cellular protein synthesis is inhibited when as little as 20% of theeIF-2a molecules are phosphorylated. Significant inhibition of proteinsynthesis reduces expression of ssRNA that is introduced, therebycounteracting the desired outcome for which the ssRNA was introduced inthe first place, and if protein synthesis is prolonged, the cell isweakened and, ultimately, the cell progresses toward death. IFN alsoinduces and/or activates other RNA sensors. For example, IFN induces a2′-5′-oligoadenylate synthase (2′-5′OAS)/RNase L system. The 2′-5′OASenzymes are composed of two domains that assemble in the cell to form adsRNA activation site. Upon binding to dsRNA, the 2′-5′OAS is activatedto a form that converts ATP to PPi and 2′-5′-linked oligoadenylates. Inturn, the 2′-5′-linked oligoadenylates bind to enzymatically inactiveRNase L monomers, which dimerize to form enzymatically active RNase Ldimers. The active RNase L dimers then degrade RNA in the cell, furtherdecreasing protein synthesis. Still further with respect to innateimmune recognition of RNA that is introduced into a cell (e.g., byinfection with an RNA virus), the cytoplamic or cytosolic receptorsRIG-I (encoded by retinoic acid inducible gene I) and MDA5 (encoded bymelanoma differentiation associated gene-5) have important roles. RIG-Iappears to recognize and bind at least three elements of RNA structure:(i) it recognizes and preferentially binds blunt-ended short dsRNA withor without a 5-triphosphate group; (ii) it specifically recognizes andbinds 5′-triphosphate groups on ssRNA or double-stranded RNA, but doesnot recognize or binds those RNAs if they are 5′-capped; and (iii) itrecognizes and binds RNAs with polyuridine sequences (Kato H et al.,2008; Homung V et al. 2006; Jiang et al. 2011; Pichlmair A et al., 2006;Saito T et al., 2008; Schlee M et al., 2009; Uzri D and Gehrke L, 2009).On the other hand, MDA-5 specifically recognizes and binds to longdsRNA, rather than short dsRNA like RIG-I). Further, Zust et al. (2011)showed that MDA-5 also mediates sensing of ssRNA that lacks a 5′ capwith a cap1 structure; thus, a mutant corona virus that lacked2′-O-methyltransferase activity and made ssRNA had a cap0 structureresulted in MDA-5-dependent induction of type I interferons in mice,whereas wild-type corona virus that had 2′-O-methyltransferase activityand made ssRNA that had a cap1 structure did not result induction oftype I interferons, and the induction of type I interferon by2′-O-methyltransferase-deficient viruses was dependent on cytoplasmicMDA5. Upon detection of RNA exhibiting one or more of the elements theyrecognize, the cytoplasmic RNA sensors RIG I or MDA-5 then initiatesignaling cascades that induce the expression of cytokines, includingtype I interferons (IFN-α and IFN-β), which are secreted by theactivated cells to transmit danger signals to neighboring cells. Thesedanger signals are transmitted by binding of the secreted interferons totype I interferon receptors on the surfaces of the neighboring cells andthe activated type I interferon receptor (IFNAR) triggers a signalingpathway consisting of Jak and STAT transcription factors, therebyactivating expression of numerous interferon-stimulated genes.Furthermore, it is known that dsRNA also binds to other cellular RNAsensors that result in induction and/or activation of many genes. Forexample, dsRNA directly or indirectly induces transcription factors ofthe IRF family, particularly IRF 1, IRF 3, IRF 5 and IRF 7, which, inturn, induce production of more type I IFN and type I IFN induces aboutone thousand IFN-stimulated genes. Induction of these and other RNAsensors and innate immune response pathways (e.g., toll-like receptors(TLRs) TLR3, TLR7, and TLR8; retinoic acid inducible gene I (RIG-I);melanoma differentiation associated gene-5 (MDA5); and possibly thehelicase LGP2), result in inhibition of protein synthesis in theaffected cell and, ultimately, dsRNA-induced apoptosis via deathreceptor signaling, including caspase-8 activation. PKR, RNase L, IRF3and c-Jun N-terminal kinase have been reported to be components of thedsRNA-activated pro-apoptotic pathways. Thus, it is important that RNAmolecules introduced into living human and animal cells must avoidinducing and activating the numerous RNA sensors and mechanisms thatprotect them against pathogens comprising RNA. Conceivably, RNApreparations containing even minute amounts of dsRNA can trigger anundesirable innate immune response in vivo, such as an interferon(IFN)-induced and/or IFN-activated response, which leads to translationsuppression and cell death in vivo (Yang S et al., 2001; Wianny F andZemicka-Goetz M, 2000).

In some EXAMPLES herein describing embodiments of the methods comprisingreprogramming of cells that exhibited a first differentiated state orphenotype to cells that exhibited a second differentiated state orphenotype, qRT-PCR was performed on total cellular RNA purified fromcells transfected with mRNA reprogramming mixes in order to quantify thelevels mRNAs in those cells which would be indicative of induction ofRNA sensors or innate immune system response genes. For example, incertain experiments, qRT-PCR was performed using primer pairs to amplifylevels of expression for mRNAs encoding IFNB, RIG1, OAS3, and IFIT1 incells that were being reprogrammed using mRNA mixes encoding the iPSCreprogramming factors, wherein said mRNAs were either treated using theRNase III treatment method described herein or purified by HPLC. Forexample, in these qRT-PCR assays, the expression levels of the mRNAsencoding IFNB, RIG1, OAS3, and IFIT1, normalized for expression levelsof certain housekeeping genes, were low and the mRNA levels for thesegenes in the cells being transfected with RNase III-treated mRNAreprogramming mix were similar to the mRNA levels for these genes in thecells being transfected with the same mRNA reprogramming mix that wasHPLC purified. Thus, in some embodiments, activation or induction ofexpression of one or more RNA sensor or innate immune response genes isdetected, assayed, measured and/or quantified by detecting, assaying,measuring and/or quantifying the levels or relative levels of mRNAexpressed in the cells by PCR or qRT-PCR (e.g., after introducing ofmRNA reprogramming mixes into said cells).

In some embodiments, a composition, system, kit or method of the presentinvention comprises or uses a composition comprising invitro-synthesized ssRNA or mRNA synthesized using an RNA amplificationreaction, An “RNA amplification reaction” or an “RNA amplificationmethod” means a method for increasing the amount of RNA corresponding toone or multiple desired RNA sequences in a sample. For example, in someembodiments, the RNA amplification method comprises: (a) synthesizingfirst-strand cDNA complementary to the one or more desired RNA moleculesby RNA-dependent DNA polymerase or reverse transcriptase extension ofone or more primers that anneal to the desired RNA molecules; (b)synthesizing double-stranded cDNA from the first-strand cDNA using aprocess wherein a functional RNA polymerase promoter is joined thereto;and (c) contacting the double-stranded cDNA with an RNA polymerase thatbinds to said promoter under transcription conditions whereby RNAcorresponding to the one or more desired RNA molecules is obtained.Unless otherwise stated related to a specific embodiment of theinvention, an RNA amplification reaction according to the presentinvention means a sense RNA amplification reaction, meaning an RNAamplification reaction that synthesizes sense RNA (e.g., RNA having thesame sequence as an mRNA or other primary RNA transcript, rather thanthe complement of that sequence). Sense RNA amplification reactionsknown in the art, which are encompassed within this definition include,but are not limited to, the methods which synthesize sense RNA describedin Ozawa et al. (2006) and in U.S. Patent Application Nos. 20090053775;20050153333; 20030186237; 20040197802; and 20040171041. The RNAamplification method described in U.S. Patent Application No.20090053775 (now U.S. Pat. Nos. 8,039,214 and 8,329,887) by Dahl andSooknanan is a preferred method for obtaining amplified RNA derived fromone or more cells, which amplified RNA is then used to make mRNA for usein the methods of the present invention.

As used herein, “RNase III” when used herein with respect to a method,composition, kit or system of the invention means an RNase III familyendoRNase. In preferred embodiments of the methods, compositions or kitscomprising RNase III or use or methods of use thereof, the RNase IIIbinds and digests dsRNA containing a minimum of two turns of the A-formdouble helix, (approximately 20 bp), but not ssRNA, to small dsRNAoligoribonucleotides having a size of about 12 to 15 bp in length. Insome preferred embodiments, the RNase III is a class I RNase III. Insome preferred embodiments, the RNase III is derived from a microbialsource (e.g., a prokaryotic source). In one preferred embodiment, theRNase III is an enzyme derived from E. coli, or a functional fragment orvariant enzyme thereof. In some other embodiments, the RNase IIIgenerates dsRNA oligoribonucleotides less than about 30 nucleotides inlength. In preferred embodiments, the RNase III exhibits at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%,approximately 96%, approximately 97%, approximately 98%, approximately99%, or approximately 100% amino acid sequence identity with E. coliRNase III. However, RNase III, which is sometimes abbreviated as “RIII”herein, can be any double-strand-specific endoribonuclease (endoRNase)that digests dsRNA, but not ssRNA, to a similar extent as Escherichiacoli RNase III, either under approximately similar reaction conditionsas described herein, or under other reaction conditions which areoptimal for another particular highly purified RNase III that lacksendoribonuclease and exoribonuclease activity on ssRNA. Optimal reactionconditions for other RNase III family enzymes can be identified by usingthe novel RNA substrate comprising both single-stranded anddouble-stranded portions developed herein (FIG. 1); this substrateenables rapid, accurate and precise assay and optimization ofdsRNA-specific RNase activity and specificity of digestion of dsRNAversus ssRNA. As discussed herein, this substrate was used by theapplicants to develop the RNase III treatment method of the presentinvention, which much more completely digests dsRNA contaminants in RNAsamples comprising primarily ssRNA, while better preserving the ssRNAintegrity than the RNase III assay method conditions by Robertson andco-workers in their 1968 paper (Robertson, H D et al, 1968) and usedcontinuously and universally since that time (e.g., Robertson H D andHunter T, 1975; Robertson HD, 1982; Mellits K H et al., 1990, NicholsonA W, 1996, Pe'ery T and Mathews M B. 1997).

An “RNA-dependent DNA polymerase” or “reverse transcriptase” is anenzyme that is capable of extending the 3′-end of a nucleic acid that isannealed to an RNA template to synthesize DNA that is complementary tothe template (“complementary DNA” or “cDNA”). The 3′-end of the nucleicacid that is extended can be the 3′-end of the same RNA template, inwhich case cDNA synthesis is primed intramolecularly, or the 3′-end ofthe nucleic acid that is extended can be the 3′-end of another nucleicacid that is different from the RNA template and that is annealed to theRNA template, in which case cDNA synthesis is primed intermolecularly.All known reverse transcriptases also have the ability to make acomplementary DNA copy from a DNA template; thus, they are both RNA- andDNA-dependent DNA polymerases.

As used herein, a “single-strand-specific DNase” means a DNase thatspecifically digests single-stranded DNA, but that does not digestsingle-stranded RNA or RNA or DNA that is annealed to or complexed withcomplementary RNA or DNA, whether said complementary RNA or DNA is partof another nucleic acid molecule (e.g., by intermolecular base-pairing)or a portion of the same nucleic acid molecule (e.g., by intramolecularbase-pairing). The single-strand-specific DNase can be an endonucleaseor an exonuclease, so long as it is active in specifically digestingsingle-stranded DNA to monomers or short oligodeoxyribonucleotides. Inpreferred embodiments, the products of digestion using thesingle-strand-specific DNase do not serve as primers in the presence ofa single-stranded nucleic acid molecule that is capable of serving as atemplate under the reaction conditions used in the method. ExonucleaseI, exonuclease VII, and Rec J exonuclease are exemplarysingle-strand-specific DNases.

As used herein, a “single-strand-specific RNase” means an RNase thatspecifically digests single-stranded RNA, but that does not digestsingle-stranded DNA or RNA or DNA that is annealed to or complexed withcomplementary RNA or DNA, whether said complementary RNA or DNA is partof another nucleic acid molecule (e.g., by intermolecular base-pairing)or a portion of the same nucleic acid molecule (e.g., by intramolecularbasepairing). The single-strand-specific RNase can be an endonuclease oran exonuclease, so long as it is active in specifically digestingsingle-stranded RNA to monomers or short oligoribonucleotides that donot serve as primers in the presence of a single-stranded nucleic acidmolecule that is capable of serving as a template under the reactionconditions used in the method. E. coli RNase I is an exemplarysingle-strand-specific RNase.

A “poly-A polymerase” or “poly(A) polymerase” (“PAP”) means atemplate-independent RNA polymerase found in most eukaryotes,prokaryotes, and eukaryotic viruses that selectively uses ATP toincorporate AMP residues to 3′-hydroxylated ends of RNA. Since PAPenzymes that have been studied from plants, animals, bacteria andviruses all catalyze the same overall reaction (Edmonds 1990) are highlyconserved structurally (Gershon 2000) and lack intrinsic specificity forparticular sequences or sizes of RNA molecules if the PAP is separatedfrom proteins that recognize AAUAAA polyadenylation signals (Wilusz andShenk 1988), purified wild-type and recombinant PAP enzymes from any ofa variety of sources can be used for the present invention. For example,in some embodiments of compositions, kits, or methods of the invention,a “polyadenylated” or “poly(A)-tailed” ssRNA or mRNA is made using awild-type or recombinant Saccharomyces (e.g., from S. cerevisiae) PAPenzyme or Escherichia (e.g., E. coli) PAP enzyme. In some embodiments,the polyadenylated or poly(A)-tailed ssRNA or mRNA comprises or consistsof one or more ssRNAs or mRNAs containing nucleosides comprising one ormore modified nucleic acid bases that results in reduced induction oractivation of an RNA sensor or innate immune response mechanism comparedto nucleosides comprising canonical GAUC nucleic acid bases (e.g., eachof which encodes a reprogramming factor, e.g., an iPS cell inductionfactor). In other embodiments, the polyadenylated or poly(A)-tailedssRNA or mRNA comprises nucleosides comprising only canonical nucleicacid bases and does not comprise nucleosides comprising one or moremodified nucleic acid bases that results in reduced induction oractivation of an RNA sensor or innate immune response mechanism comparedto GAUC bases.

In some preferred embodiments of compositions, kits, or methods of theinvention, the ssRNA or mRNA comprises or consists of invitro-synthesized (or in vitro-transcribed) ssRNA or mRNA (or ssRNA ormRNA molecules), each of which “encodes” (or “exhibits a coding region”or (coding sequence” (“cds”) or “exhibits an open reading frame” (“ORF”)of a particular protein or polypeptide (e.g., a particular protein orpolypeptide reprogramming factor), meaning that each ssRNA or mRNAexhibits a linear array of codon triplets defined by the sequence ofnucleotides that extends from the translation initiation codon to thetranslation termination codon for one particular protein or polypeptide.In addition to exhibiting the ORF of a particular protein, each ssRNAmolecule may also exhibit other sequences 5′-of or 3′-of the ORF whichare referred to as “5′ or 3′ untranslated regions” or “5′ or 3′ UTRs,”which may serve different functions. For example, in some preferredembodiments, the 5′ UTR comprises a Kozak consensus or Kozak sequence. AKozak sequence is a sequence which occurs on eukaryotic mRNA and has theconsensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine)three bases upstream of the start codon (AUG), which is followed byanother ‘G’ (Kozak M, 1987). The Kozak consensus sequence plays animportant role in the initiation of the translation process.

In some preferred embodiments of compositions, kits, or methods of theinvention, the one or more in vitro-synthesized ssRNAs or mRNAs and/orpurified ssRNAs or mRNAs exhibit at least one heterologous 5′ UTRsequence, Kozak sequence, IRES sequence, or 3′ UTR sequence that resultsin greater translation into the encoded protein when said respectivessRNAs are introduced into eukaryotic cells compared to the same ssRNAsthat do not exhibit said respective 5′ UTR sequence, Kozak sequence,IRES sequence, or 3′ UTR sequence. In some particular preferredembodiments, the 5′ UTR or 3′ UTR is a sequence exhibited by a Xenopusor human alpha- (α-) globin or beta- (β-) globin mRNA, or wherein the 5′UTR is a sequence exhibited by tobacco etch virus (TEV) RNA.

In some preferred embodiments of a composition, kit or method of theinvention, the RNA composition comprising ssRNA or mRNA is treated orpurified (e.g. by treating with a dsRNA-specific RNase (e.g., adsRNA-specific endoribonuclease (endoRNase), e.g., wild-type orrecombinant RNase III or an active fragment or variant thereof),treating with a dsRNA-specific antibody, and/or purifying byphenol-chloroform extraction, ammonium acetate precipitation, orchromatography, including by HPLC) (e.g., prior to use of saidcomposition comprising ssRNA or mRNA in the method of the invention forreprogramming). In some embodiments of the compositions, kits or methodsof the invention, the treated or purified ssRNA or mRNA exhibits a 5′cap comprising 7-methylguanine or an anti-reverse cap analog (ARCA,including an ARCA with a thio group in the triphosphate bridge). In someembodiments, the treated ssRNAs or purified ssRNA or mRNA furthercomprises a 5′ cap that has a cap1 structure, wherein the 2′ hydroxyl ofthe ribose in the 5′ penultimate nucleotide is methylated. In someembodiments, wherein the treated or purified ssRNA or mRNA exhibits a 5′cap, the one or more in vitro-synthesized ssRNAs or mRNAs used for saidtreating and/or purifying exhibits the 5′ cap (i.e., prior to saidtreating or purifying). Thus, in some embodiments, the one or more invitro-synthesized ssRNAs or mRNAs used for said treating and/orpurifying comprise capped ssRNAs or mRNAs. In some of these embodiments,the one or more in vitro-synthesized ssRNA molecules that exhibit the 5′cap were synthesized prior to said treating and/or purifying: (i)co-transcriptionally by incorporation of a cap analog (e.g., ananti-reverse cap analog or ARCA, or e.g., a thio-ARCA)) during in vitrotranscription (e.g., using the MESSAGEMAX™ T7 ARCA-capped messagetranscription kit or the INCOGNITO™ T7 ARCA 5^(m)C- and Ψ-RNAtranscription kit, CELLSCRIPT, INC., Madison, Wis., USA); or (ii)post-transcriptionally by incubating in vitro-transcribed ssRNAmolecules with a capping enzyme system comprising RNA guanyltransferaseunder conditions wherein the in vitro-transcribed ssRNA molecules are5′-capped, including wherein the capping enzyme system results inmethylation of the 2′ hydroxyl of the ribose in the 5′ penultimatenucleotide (e.g., using T7 mSCRIPT™ standard mRNA production system, orusing a separate in vitro transcription system, such as the T7-SCRIBE™standard RNA IVT kit, the INCOGNITO™ T7 Ψ-RNA transcription kit, or theINCOGNITO™ T7 5mC- and Ψ-RNA transcription kit to obtain ssRNA, and theSCRIPTCAP™ m⁷G capping system to obtain cap0 RNA (all from CELLSCRIPT,INC.); in some preferred embodiments, the capping enzyme system furtherresults in methylation of the 2′ hydroxyl of the ribose in the 5′penultimate nucleotide to generate cap1 RNA, and another step forsynthesizing said in vitro-synthesized ssRNA or mRNA comprises:incubating the in vitro-transcribed ssRNA or mRNA with RNA2′-O-methyltransferase (e.g., using the SCRIPTCAP™2′-O-methyltransferase kit, CELLSCRIPT, INC.).

In some preferred embodiments of a composition, kit or method of theinvention, wherein the treated and/or purified ssRNA or mRNA exhibits a5′ cap, the one or more in vitro-synthesized ssRNAs are uncapped; insome embodiments, prior to use of the ssRNAs or mRNAs in a method forreprogramming, another step for synthesizing said in vitro-synthesizedssRNAs or mRNAs comprises: post-transcriptionally capping the treatedand/or purified ssRNAs to generate 5′ capped treated and/or 5′ cappedpurified ssRNAs. In some preferred embodiments, said capping comprisescapping with both a capping enzyme system comprising RNAguanyltransferase and 2′-O-methyltransferase. In some embodiments, saidpost-transcriptional capping of the treated and/or purified ssRNAs isperformed as described above and/or in the product literature providedwith the SCRIPTCAP™ m⁷G Capping System, the SCRIPTCAP™2′-O-methyltransferase kit, or the T7 mSCRIPT™ standard mRNA productionsystem with respect to the capping enzyme system components (all fromCELLSCRIPT, INC., Madison, Wis., USA).

In some preferred embodiments of a composition, kit, or method of theinvention, the one or more in vitro-synthesized ssRNAs or mRNAs used forsaid treating are significantly free of uncapped RNAs that exhibit a5′-triphosphate group (which are considered to be one type of“contaminant RNA molecules” herein). In some preferred embodiments, theRNA composition comprising treated and/or purified ssRNA or mRNA issignificantly free of uncapped RNAs that exhibit a 5′-triphosphategroup. In certain embodiments, the one or more in vitro-synthesizedssRNAs used for said treating, the treated ssRNAs, and/or the purifiedssRNAs consist of a population of ssRNA molecules having: (i) greaterthan 90% capped ssRNA molecules; (ii) greater than 95% capped ssRNAmolecules; (iii) greater than 98% capped ssRNA molecules (iv) greaterthan 99% capped ssRNA molecules; or (v) greater than 99.9% capped ssRNAmolecules. In some embodiments wherein the population of ssRNA moleculesalso comprises contaminant uncapped RNA molecules that exhibit a5′-triphosphate group, prior to using said RNA composition comprisingsaid ssRNA molecules for reprogramming, the method further comprises:incubating the one or more in vitro-synthesized ssRNAs used for saidtreating, or the treated ssRNAs or the purified ssRNAs generated fromthe method with at least one enzyme to remove the triphosphate groupsfrom contaminating uncapped ssRNAs. In some embodiments, the at leastone enzyme is an alkaline phosphatase (e.g., NTPhosphatase™, epicentretechnologies, Madison, Wis., USA) or with RNA 5′ polyphosphatase(epicentre technologies); in some embodiments wherein the at least oneenzyme is RNA 5′ polyphosphatase, said ssRNA molecules for reprogrammingare further incubated with TERMINATOR™ 5′-phosphate-dependent nuclease(epicentre technologies) or Xm1 exoribonuclease (e.g., fromSaccharomyces cerevisae) to digest said uncapped RNA from which the5′-triphosphate group has been removed. These methods for incubatingwith alkaline phosphatase or with RNA 5′ polyphosphatase and,optionally, also with TERMINATOR™ 5′-phosphate-dependent nuclease or Xm1exoribonuclease, are particularly useful to remove uncapped ssRNAs fromcapped ssRNAs that were made by co-transcriptional capping byincorporating a cap analog during an in vitro transcription reaction.

“Stem cells” herein mean cells that have three general properties whichmake them different from other kinds of cells in the body: (1) they arecapable of long-term self-renewal, meaning that, unlike specialized ordifferentiated cells which do not normally replicate themselves, theycan proliferate by division of single cells into two daughter cellswhich are identical to the mother cell for long periods; (2) they areunspecialized, meaning they do not have any cell-specific structures forperforming specialized functions; and (3) they can give rise tospecialized cell types by a process called “differentiation.”Information about stem cells is available on a National Institutes ofHealth website dedicated to that purpose (http://followed by“stemcells.nih.gov/info”).

“Pluripotent stem cells” herein mean cells that can give rise to anytype of cell in the body except those needed to support and develop afetus in the womb.

Different methods are used to assay or evaluate a cell with respect toits pluripotent status. For example, the embryoid body spontaneousdifferentiation assay (e.g., see EXAMPLES) is sometimes used to evaluatethe capability of cells or a cell line to differentiate into cellrepresenting all three germ layers. Another method that is used is toperform fluorescent immunostaining assays using fluorescent antibodiesthat bind to proteins that are known to be expressed in pluripotentcells (e.g., see EXAMPLES). Still another type of assay that can beperformed to evaluate pluripotency is quantitative reverse transcriptionpolymerase chain reaction or qRT-PCR, sometimes simply called “qPCR” Inthese assays, qPCR is performed to quantify the relative level ofexpression of certain mRNAs encoding proteins that are known to beexpressed or expressed at certain relative levels in pluripotent cellscompared to the expression levels of certain housekeeping genes whichare approximately constitutively expressed. Examples of pluripotencymRNAs which can be assayed by qRT-PCR include mRNAs encoding CRIPTO,GDF3, NANOG, OCT4 and REX1 (e.g., see EXAMPLES). For example, in someqRT-PCR assays performed using total cellular RNA isolated from 6different IPSC lines generated in reprogramming experiments describedherein, significantly higher levels of mRNAs encoding CRIPTO, GDF3,NANOG, OCT4 and REX1 were measured in the iPSC lines generated from mRNAreprogramming with RNase III-treated or HPLC-purified mRNA than wasmeasured in the original human primary foreskin BJ fibroblast cells andthe feeder cells (NUFFs) used in those experiments.

“Induced pluripotent stem cells” (“iPSCs”) herein mean adult cells thathave been genetically induced or reprogrammed to an embryonic stemcell-like state by being forced to express genes and factors importantfor maintaining certain defining properties of embryonic stem cells,such as expression of embryonic stem cell markers and being capable ofdifferentiation into cells from all three germ layers.

An “iPSC line” herein means stem cells derived from a single iPSC colonythat maintain these certain defining properties of embryonic stem cellsupon repeated propagation in culture.

A “reprogramming factor” means a protein, polypeptide, or otherbiomolecule that, when used alone or in combination with other factorsor conditions, causes a change in the state of differentiation of a cellin which the reprogramming factor is introduced or expressed. In somepreferred embodiments of the methods of the present invention, thereprogramming factor is a protein or polypeptide that is encoded by anmRNA that is introduced into a cell, thereby generating a cell thatexhibits a changed state of differentiation compared to the cell inwhich the mRNA was introduced. In some preferred embodiments of themethods of the present invention, the reprogramming factor is atranscription factor. One embodiment of a reprogramming factor used in amethod of the present invention is an “iPS induction factor,” meaning aprotein, peptide, or other biomolecule that, when used alone or incombination with other factors or conditions, causes a change in thestate of differentiation of a cell into which the iPS cell inductionfactor is introduced and/or expressed to an induced pluripotent stemcell (or iPSC).

An “mRNA reprogramming factor” means an mRNA that encodes areprogramming factor consisting of a protein or polypeptide. An “mRNAiPSC induction factor” is one embodiment of an mRNA reprogramming factorand means an mRNA that encodes and iPSC induction factor.

The terms “mRNA reprogramming mix” or “mRNA reprogramming factor mix” or“ssRNA reprogramming mix” or “ssRNA reprogramming factor mix” are usedinterchangeably herein and mean a mixture of mRNAs encoding differentreprogramming factors, each consisting of a protein or polypeptide.

Similarly, the terms “mRNA iPSC reprogramming mix” or “mRNA iPSCinduction factor mix” or “ssRNA iPSC reprogramming mix” or “ssRNA iPSCinduction mix” or “ssRNA iPSC induction factor mix” are usedinterchangeably herein and mean a mixture of mRNAs encoding differentiPSC induction factors, each consisting of a protein or polypeptide. An“iPS cell induction factor” or “iPSC induction factor” is a protein,polypeptide, or other biomolecule that, when used alone or incombination with other reprogramming factors, causes the generation of adedifferentiated cell or iPS cells from somatic cells. Examples of iPScell induction factors include OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28.iPS cell induction factors include full length polypeptide sequences orbiologically active fragments thereof. Likewise an mRNA encoding an iPScell induction factor may encode a full length polypeptide orbiologically active fragments thereof. The DNA template sequences formRNAs encoding exemplary iPS induction factors are shown in SEQ ID NOS:2-10. In certain embodiments, the present invention employs the DNAtemplate sequences or similar sequences shown in these SEQ ID NOS,including DNA template sequences encoding ssRNAs or mRNAs molecules thatadditionally comprise, joined to these ssRNA or mRNA sequences,oligoribonucleotides which exhibit any of the 5′ and 3′ UTR sequences,Kozak sequences, IRES sequences, cap nucleotides, and/or poly(A)sequences used in the experiments described herein (e.g., as shown inSEQ ID NO. 1), or other UTR or other sequences which are generally knownin the art or discovered in the future which can be used in place ofthose used herein by joining them to these protein-coding mRNA sequencesfor the purpose of optimizing translation of the respective mRNAmolecules in the cells and improving their stability in the cell inorder to accomplish the methods described herein.

“Differentiation” or “cellular differentiation” means the naturallyoccurring biological process by which a cell that exhibits a lessspecialized state of differentiation or cell type (e.g., a fertilizedegg cell, a cell in an embryo, or a cell in a eukaryotic organism)becomes a cell that exhibits a more specialized state of differentiationor cell type. Scientists, including biologists, cell biologists,immunologists, and embryologists, use a variety of methods and criteriato define, describe, or categorize different cells according to their“cell type,” “differentiated state,” or “state of differentiation.” Ingeneral, a cell is defined, described, or categorized with respect toits “cell type,” “differentiated state,” or “state of differentiation”based on one or more phenotypes exhibited by that cell, which phenotypescan include shape, a biochemical or metabolic activity or function, thepresence of certain biomolecules in the cell (e.g., based on stains thatreact with specific biomolecules), or on the cell (e.g., based onbinding of one or more antibodies that react with specific biomoleculeson the cell surface). For example, in some embodiments, different celltypes are identified and sorted using a cell sorter orfluorescent-activated cell sorter (FACS) instrument. “Differentiation”or “cellular differentiation” can also occur to cells in culture. Asused herein, it will be understood that the difference between a cellthat exhibits a first state of differentiation, differentiated state,cell type or phenotype and a cell that exhibits a second state ofdifferentiation, differentiated state, cell type or phenotype state canrange from a difference in the relative expression of a single proteinto differences in the expression of multiple proteins; thus, in someembodiments, the cell that exhibits a second state of differentiation,differentiated state, cell type or phenotype differs from the cell thatexhibits a first state of differentiation, differentiated state, celltype or phenotype because the cell that exhibits a second state ofdifferentiation, differentiated state, cell type or phenotype expressesa protein or multiple proteins that is or are encoded by mRNA moleculethat are introduced into the cell that exhibits the first state ofdifferentiation, differentiated state, cell type or phenotype, whereasin other embodiments, the cell that exhibits a second state ofdifferentiation, differentiated state, cell type or phenotype differsfrom the cell that exhibits a first state of differentiation,differentiated state, cell type or phenotype because the cell thatexhibits a second state of differentiation, differentiated state, celltype or phenotype expresses one or more proteins that are induced bymRNA molecules that are introduced into the cell that exhibits a firststate of differentiation, differentiated state, cell type or phenotype,even though one or more of those proteins may not be encoded by saidmRNA molecules that are introduced into the cell that exhibits a firststate of differentiation, differentiated state, cell type or phenotype.

The term “reprogramming” as used herein means an induced or anon-naturally-occurring process of changing the state of differentiationor phenotype of a cell in response to delivery of one or morereprogramming factors into the cell, directly (e.g., by delivery ofprotein or polypeptide reprogramming factors into the cell) orindirectly (e.g., by delivery of the purified RNA preparation of thepresent invention which comprises one or more mRNA molecules, each ofwhich encodes a reprogramming factor) and maintaining the cells underconditions (e.g., medium, temperature, oxygen and CO₂ levels, matrix,growth factors, cytokines, cytokine inhibitors, and other environmentalconditions) that are conducive for differentiation. The term“reprogramming” when used herein is not intended to mean or refer to aspecific direction or path of differentiation (e.g., from a lessspecialized cell type to a more specialized cell type) and does notexclude processes that proceed in a direction or path of differentiationthan what is normally observed in nature. Thus, in different embodimentsof the present invention, “reprogramming” means and includes any and allof the following:

(1) “Dedifferentiation”, meaning a process by which a cell that exhibitsa more specialized state of differentiation or cell type (e.g., amammalian fibroblast, a keratinocyte, a muscle cell, or a neural cell)becomes a cell that exhibits a less specialized state of differentiationor cell type (e.g., a dedifferentiated cell or an iPS cell);

(2) “Transdifferentiation”, meaning a process by which a cell thatexhibits a more specialized state of differentiation or cell type (e.g.,a mammalian fibroblast, a keratinocyte, or a neural cell) becomes a cellthat exhibits another more specialized state of differentiation or celltype (e.g., from a fibroblast or keratinocyte to a muscle cell); and

(3) “Redifferentiation” or “expected differentiation” or naturaldifferentiation”, meaning a process by which a cell that exhibits anyparticular state of differentiation or cell type becomes a cell thatexhibits another state of differentiation or cell type as would beexpected in nature if the cell was present in its natural place andenvironment (e.g., in an embryo or an organism), whether said processoccurs in vivo in an organism or in culture (e.g., in response to one ormore reprogramming factors).

A “double-strand-specific RNase” herein means an exoribonuclease orendoribonuclease that digests dsRNA, but not ssRNA, tomonoribonucleotides or small oligoribonucleotides (e.g., tooligoribonucleotides having a size less than about 30 nucleotides), butthat does not digest ssRNA.

A “dsRNA-specific 3′-to-5′ exoribonuclease” means and includes anyexoribonuclease that digests dsRNA, but not ssRNA, in a 3′-to-5′direction, starting from 3′-ends that are annealed to a complementaryRNA.

By a “purified or treated RNA composition” (which, when used in a methodof the present invention, is sometimes referred to only as “ssRNA”,“mRNA” or an “RNA composition”), we mean a composition that comprises orconsists of one or more treated or purified ssRNAs or mRNAs that is orare substantially free, virtually free, essentially free, practicallyfree, extremely free or absolutely free of dsRNA molecules as definedherein.

For example, an RNA composition or ssRNA or mRNA that is substantiallyfree of dsRNA molecules would contain less than five nanograms of dsRNAof a size greater than about 40 basepairs in length per microgram ofRNA.

For example, an RNA composition or ssRNA or mRNA that is virtually freeof dsRNA molecules would contain less than one nanogram of dsRNA of asize greater than about 40 basepairs in length per microgram of RNA.

For example, an RNA composition or ssRNA or mRNA that is essentiallyfree of dsRNA molecules would contain less than 0.5 nanogram of dsRNA ofa size greater than about 40 basepairs in length per microgram of RNA.

For example, an RNA composition or ssRNA or mRNA that is practicallyfree of dsRNA molecules would contain less than 100 picograms of dsRNAof a size greater than about 40 basepairs in length per microgram ofRNA.

For example, an RNA composition or ssRNA or mRNA that is extremely freeof dsRNA molecules would contain less than 10 picograms of dsRNA of asize greater than about 40 basepairs in length per microgram of RNA.

For example, an RNA composition or ssRNA or mRNA that is absolutely freeof dsRNA molecules would contain less than 2 picograms of dsRNA of asize greater than about 40 basepairs in length per microgram of RNA.

Similarly, it will also be understood herein that an “RNA composition”or a “ssRNA composition” or “ssRNA molecules” or “mRNA” or a“reprogramming mix” or a “ssRNA reprogramming mix” or an “mRNAreprogramming mix” or a “ssRNA iPSC reprogramming mix” or an “mRNA iPSCreprogramming mix” or a “ssRNA iPSC induction mix” or an “mRNAreprogramming factor mix” or “a mixture of reprogramming factors” or “amixture of iPSC induction factors” (or the like) that is or are“practically free,” “extremely free,” or “absolutely free” of dsRNAherein means that less than 100 picograms, less than 10 picograms, orless than 2 picograms, respectively of dsRNA of a size greater thanabout 40 basepairs is present per microgram of RNA in said RNAcomposition, ssRNA composition, ssRNA molecules, ssRNA, mRNA, ssRNA iPSCreprogramming mix, mRNA iPSC reprogramming mix, mRNA reprogrammingfactor mix, mixture of reprogramming factors, mixture of iPSC inductionfactors, or the like. In some embodiments, the amount of dsRNA isdetermined using a dot blot assay wherein the amount of dsRNA isquantified by immunoassay using the J2 dsRNA-specific antibody (English& Scientific Consulting, Szirák, Hungary) (e.g., compared to knownamounts of dsRNA standards spotted on nylon membranes in parallel assaysusing methods identical to or equivalent to those described herein). Inother embodiments, the amount of dsRNA is determined by another method,such as by comparative HPLC using known standards.

DESCRIPTION OF THE INVENTION

In some embodiments, the present invention relates to compositions andmethods for reprogramming somatic cells to pluripotent stem cells. Forexample, the present invention provides RNA compositions comprisingssRNA (e.g., mRNA molecules) and their use to reprogram human or animal(e.g., mammalian) somatic cells into pluripotent stem cells. Forexample, in some embodiments the invention providespseudouridine-modified (Ψ-modified) and/or 5-methylcytidine-modified(m⁵C-modified) ssRNA molecules that are at least practically free ofdsRNA molecules, more preferably, at least extremely free of dsRNAmolecules, and most preferably, absolutely free of dsRNA molecules andthat encode reprogramming factors.

Experiments conducted during the development of embodiments of thepresent invention demonstrated that mRNA molecules can be administeredto cells and induce a dedifferentiation process to generatededifferentiated cells—including pluripotent stem cells. Thus, thepresent invention provides compositions and methods for generatingdedifferentiated or iPS cells. Surprisingly, the administration ofsingle-stranded mRNA that is at least practically free, and preferablyat least extremely free or at least absolutely free of dsRNA can providehighly efficient generation of dedifferentiated or iPS cells.Unexpectedly and surprisingly, not only modified mRNA, such aspseudouridine- (Ψ-) and/or 5-methylcytidine-(m⁵C-) modified mRNAencoding iPS cell induction factors, but also unmodified mRNA encodingsaid iPS cell induction factors, results in highly efficient generationof dedifferentiated cells or iPS cells.

In some embodiments, the present invention provides methods fordedifferentiating a somatic cell comprising: introducing mRNA encodingone or more iPSC induction factors into a somatic cell to generate adedifferentiated cell.

In some embodiments, the present invention provides methods fordedifferentiating a somatic cell comprising: introducing a ssRNAcomposition comprising mRNA molecules encoding one or more iPSCinduction factors into a somatic cell and maintaining the cell underconditions wherein the cell is viable and the mRNA that is introducedinto the cell is expressed in sufficient amount and for sufficient timeto generate a dedifferentiated cell. In some preferred embodiments, thededifferentiated cell is an induced pluripotent stem cell (iPSC). Insome embodiments of the methods of the present invention forreprogramming a cell from a first state of differentiation or phenotypeto a second state of differentiation or phenotype comprising an iPScell, or of compositions, systems or kits performing said method, or ofcompositions that result from use of said methods, the iPS cellexpresses the inner cell mass-specific marker NANOG (which is one markerused to assay whether a dedifferentiated cell is an iPS cell, e.g., seeGanzalez et al. 2009, and Huangfu et al. 2008). In some otherembodiments of the methods of the present invention for reprogramming acell from a first state of differentiation or phenotype to a secondstate of differentiation or phenotype comprising an iPS cell, or ofcompositions, systems or kits performing said method, or of compositionsthat result from use of said methods, the iPS cell expresses TRA-1-60(which is considered to be a more stringent marker of fully reprogrammediPS cells used to assay whether a dedifferentiated cell is an iPS cell,e.g., see Chan et al. 2009). In preferred embodiments of this method orof compositions, systems or kits performing said method, or ofcompositions that result from use of said methods, the RNA compositioncomprising ssRNA or mRNA used for said introducing is substantiallyfree, virtually free, essentially free, practically free, extremely freeor absolutely free of dsRNA.

In some embodiments, the present invention provides methods for changingthe state of differentiation (or differentiated state) of a eukaryoticcell (e.g., a human or animal cell) comprising: introducing a ssRNAcomposition comprising mRNA molecules encoding one or more reprogrammingfactors into a cell and maintaining the cell under conditions whereinthe cell is viable and the mRNA that is introduced into the cell isexpressed or translated into proteins in sufficient amounts and forsufficient time to generate a cell, wherein the cell exhibits a changedstate of differentiation compared to the cell into which the mRNA wasintroduced. In preferred embodiments of this method, the ssRNAcomposition is substantially free, virtually free, essentially free,practically free, extremely free or absolutely free of dsRNA.

In some embodiments, the present invention provides methods for changingthe state of differentiation of a eukaryotic cell (e.g., a human oranimal cell) comprising: introducing a ssRNA composition comprising mRNAencoding one or more reprogramming factors into a cell and maintainingthe cell under conditions wherein the cell is viable and the mRNA thatis introduced into the cell is expressed or translated into proteins insufficient amounts and for sufficient time to generate a cell thatexhibits a changed state of differentiation compared to the cell intowhich the mRNA was introduced. In preferred embodiments of this method,the ssRNA composition is substantially free, virtually free, essentiallyfree, practically free, extremely free or absolutely free of dsRNA. Insome embodiments, the changed state of differentiation is adedifferentiated state of differentiation compared to the cell intowhich the mRNA was introduced. For example, in some embodiments, thecell that exhibits the changed state of differentiation is a pluripotentstem cell that is dedifferentiated compared to a somatic cell into whichthe mRNA was introduced (e.g., a somatic cell that is differentiatedinto a fibroblast, a cardiomyocyte, or another differentiated celltype). In some embodiments, the cell into which the mRNA is introducedis a somatic cell of one lineage, phenotype, or function, and the cellthat exhibits the changed state of differentiation is a somatic cellthat exhibits a lineage, phenotype, or function that is different thanthat of the cell into which the mRNA was introduced; thus, in theseembodiments, the method results in transdifferentiation (Graf and Enver2009).

The methods of the invention are not limited with respect to aparticular cell into which the mRNA is introduced. In some embodimentsof any of the methods for reprogramming a eukaryotic cell, the cell intowhich the mRNA is introduced is derived from any multi-cellulareukaryote. In some preferred embodiments, the cell into which the mRNAis introduced is selected from among a human cell and an animal cell. Inother embodiments, the cell into which the mRNA is introduced isselected from among a plant and a fungal cell. In some embodiments ofany of the methods for reprogramming a eukaryotic cell, the cell intowhich the mRNA is introduced is a normal cell that is from an organismthat is free of a known disease. In some embodiments of any of themethods for reprogramming a eukaryotic cell, the cell into which themRNA is introduced is a cell from an organism that has a known disease.In some embodiments of any of the methods for reprogramming a eukaryoticcell, the cell into which the mRNA is introduced is a cell that is freeof a known pathology. In some embodiments of any of the methods forreprogramming a eukaryotic cell, the cell into which the mRNA isintroduced is a cell that exhibits a disease state or a known pathology(e.g., a cancer cell, or a pancreatic beta cell that exhibits metabolicproperties characteristic of a diabetic cell).

The invention is not limited to the use of a specific cell type (e.g.,to a specific somatic cell type) in embodiments of the methodscomprising introducing mRNA encoding one or more iPSC cell inductionfactors in order to generate a dedifferentiated cell (e.g., adedifferentiated cell or an iPS cell). Any cell that is subject todedifferentiation using iPS cell induction factors is contemplated. Suchcells include, but are not limited to, fibroblasts, keratinocytes,adipocytes, lymphocytes, T-cells, B-Cells, cells in mononuclear cordblood, buccal mucosa cells, hepatic cells, HeLa, MCF-7 or other cancercells. In some embodiments, the cells reside in vitro (e.g., in culture)or in vivo. In some embodiments, when generated in culture, a cell-freeconditioned medium (e.g., a mouse embryonic fibroblast-conditioned orMEF-conditioned medium) is used. For example, in some embodiments of themethods for reprogramming a human or mammalian cell that exhibits afirst differentiated state or phenotype to a second differentiated stateor phenotype by repeatedly or continuously introducing ssRNA or mRNAencoding one or more reprogramming factors, the cells for saidreprogramming are incubated on feeder cells during and/or after saidintroducing; in other embodiments, the cells are incubated in aMEF-conditioned medium (e.g., prepared as described by Xu et al., 2001)in the absence of feeder cells during and/or after said introducing,rather than plating them on a feeder layer. In some embodiments, thismethod is faster and more efficient than other methods for reprogrammingthan published protocols comprising transfecting cells with DNA plasmidsor lentiviral vectors encoding the same or similar reprogramming factorsin non-MEF-conditioned medium (e.g., Aoi et al. 2008). In some otherembodiments, the Stemgent PLURITON™ mRNA reprogramming medium is used toculture the somatic cells that are transfected with the purified RNAcomposition comprising ssRNA molecules that encode one or more iPS cellinduction factors until dedifferentiated or iPS cells are induced, afterwhich the dedifferentiated or iPS cells or iPSC colonies are cultured inanother medium, such as NUTRISTEM™ medium. In some other embodiments(e.g., as described in EXAMPLE 11), another medium (e.g., theFeeder-free Reprogramming Medium developed by the present inventors forreprogramming human fibroblasts to iPSCs) is used for reprogramming and,in some other embodiments (e.g., as described in EXAMPLE 11), anothermedium is used for maintenance of the iPSCs or iPSC colonies generatedfrom the reprogramming (e.g., in order to avoid redifferentiation of thededifferentiated or iPS cells or colonies into somatic cells. Asdemonstrated below, such a Feeder-free Reprogramming Medium providedenhanced and feeder-free generation of dedifferentiated or iPS cells andcolonies from human somatic cells (e.g., fibroblast cells). Theinvention is not limited, however, to the culturing conditions used. Anyculturing condition or medium now known or later identified as usefulfor the methods of the invention (e.g., to generate dedifferentiatedcells or iPS cells from somatic cells and maintain said cells) iscontemplated for use with the invention. For example, although notpreferred, in some embodiments of the method, a feeder cell layer isused instead of conditioned medium for culturing the cells that aretreated using the method.

In some embodiments of these methods, the step of introducing mRNAcomprises delivering the mRNA into the cell (e.g., a human or otheranimal somatic cell) with a transfection reagent (e.g., TRANSIT™ mRNAtransfection reagent, MirusBio, Madison, Wis.). However, the inventionis not limited by the nature of the transfection method utilized.Indeed, any transfection process known, or identified in the future thatis able to deliver mRNA molecules into cells in vitro or in vivo, iscontemplated, including methods that deliver the mRNA into cells inculture or in a life-supporting medium, whether said cells compriseisolated cells or cells comprising a eukaryotic tissue or organ, ormethods that deliver the mRNA in vivo into cells in an organism, such asa human, animal, plant or fungus. In some embodiments, the transfectionreagent comprises a lipid (e.g., liposomes, micelles, etc.). In someembodiments, the transfection reagent comprises a nanoparticle ornanotube. In some embodiments, the transfection reagent comprises acationic compound (e.g., polyethylene imine or PEI). In someembodiments, the transfection method uses an electric current to deliverthe mRNA into the cell (e.g., by electroporation).

The data presented herein shows that, with respect to the mRNAintroduced into the cell, certain amounts of the mRNAs used in theEXAMPLES described herein resulted in higher efficiency and more rapidinduction of pluripotent stem cells from the particular somatic cellsused than other amounts of mRNA. However, the methods of the presentinvention are not limited to the use of a specific amount of mRNA tointroduce into the cell. For example, in some embodiments, a total ofthree doses, with each dose comprising 18 micrograms of each of sixdifferent mRNAs, each encoding a different human reprogramming factor,was used to introduce the mRNA into approximately 3×10⁵ human fibroblastcells in a 10-cm plate (e.g., delivered using a lipid-containingtransfection reagent), although in other embodiments, higher or loweramounts of the mRNAs were used to introduce into the cells.

The invention is not limited to a particular chemical form of the mRNAused so long as the particular form of mRNA functions for its intendedapplication, although certain forms of mRNA may produce more efficientresults, which are preferred embodiments herein. In some preferredembodiments, the mRNA is polyadenylated. For example, in some preferredembodiments, the mRNA comprises a poly-A tail (e.g., a poly-A tailhaving 50-200 nucleotides, e.g., preferably 100-200, 150-200nucleotides, or greater than 150 nucleotides), although in someembodiments, a longer or a shorter poly-A tail is used. In someembodiments, the mRNA used in the methods is capped. To maximizeefficiency of expression and to minimize the innate immune response inthe cells, it is preferred that the majority, and more preferably, allor substantially all of mRNA molecules contain a cap. Thus, in somepreferred embodiments, the mRNA molecules used in the methods aresynthesized in vitro by incubating uncapped primary RNA in the presenceof with a capping enzyme system, which can result in approximately 100%of the RNA molecules being capped. In preferred embodiments, greaterthan 90%, greater than 95%, or greater than 98% of mRNA molecules arecapped. In even more preferred embodiments, greater than 99%, greaterthan 99.5%, or greater than 99.9% of the population of mRNA moleculesare capped. In preferred embodiments, the mRNA molecules used in themethods of the present invention have a cap with a cap1 structure,wherein the penultimate nucleotide with respect to the cap nucleotidehas a methyl group on the 2′-position of the ribose. For example, insome embodiments, mRNA that has cap1 structure is synthesized byincubating in vitro-transcribed RNA with SCRIPTCAP™ capping enzyme andthe SCRIPTCAP™ 2′-O-methyl-transferase enzymes (CELLSCRIPT, INC.,Madison, Wis.) or the equivalent capping enzyme components in the T7mSCRIPT™ standard mRNA production system, as described in the productliterature provided with those products (CELLSCRIPT, INC., Madison,Wis.). In some embodiments, the mRNA used in the methods of the presentinvention has a modified cap nucleotide. For example, in someembodiments, mRNA comprising a modified cap nucleotide is synthesized asdescribed in U.S. patent application Ser. No. 11/787,352 (PublicationNo. 20070281336). Thus, in some preferred embodiments, the primary RNAused in the capping enzyme reaction is synthesized by in vitrotranscription (IVT) of a DNA molecule that encodes the RNA to besynthesized. The DNA that encodes the RNA to be synthesized is joined toan RNA polymerase promoter, to which, an RNA polymerase binds andinitiates transcription therefrom. The IVT can be performed using anyRNA polymerase so long as synthesis of the template that encodes the RNAis specifically and sufficiently initiated from a respective cognate RNApolymerase promoter. In some preferred embodiments, the RNA polymeraseis selected from among T7 RNA polymerase, SP6 RNA polymerase and T3 RNApolymerase.

Thus, mRNA that has a cap1 structure, prepared by post-transcriptionalcapping of in vitro-transcribed RNA is preferred for the methodscomprising introducing purified mRNA comprising or consisting of atleast one modified ribonucleoside, which mRNA encodes at least onereprogramming factor, into a cell that exhibits a first differentiatedstate or phenotype to generate a reprogrammed cell that exhibits asecond differentiated state or phenotype. However, in some otherembodiments, capped RNA is synthesized co-transcriptionally by using adinucleotide cap analog in the IVT reaction (e.g., using an AMPLICAP™ T7Kit or a MESSAGEMAX™ T7 ARCA-CAPPED MESSAGE Transcription Kit;CELLSCRIPT, INC., Madison, Wis., USA). If capping is performedco-transcriptionally, preferably the dinucleotide cap analog is ananti-reverse cap analog (ARCA). However, use of a separate IVT reaction,followed by capping with a capping enzyme system, which results inapproximately 100% of the RNA being capped, is preferred overco-transcriptional capping, which typically results in only about 80% ofthe RNA being capped. Thus, in some preferred embodiments, a highpercentage of the mRNA molecules used in a method of the presentinvention are capped (e.g., greater than 80%, greater than 90%, greaterthan 95%, greater than 98%, greater than 99%, greater than 99.5%, orgreater than 99.9% of the population of mRNA molecules are capped). Insome preferred embodiments, the mRNA used in the methods of the presentinvention has a cap with a cap1 structure, meaning that the penultimatenucleotide with respect to the cap nucleotide has a methyl group on the2′-position of the ribose. Capped RNA synthesized co-transcriptionallyby using a dinucleotide cap analog in the IVT reaction can be convertedto mRNA that has a cap1 structure by incubating said capped RNA with anRNA 2′-O-methyltransferase enzyme (e.g., SCRIPTCAP™2′-O-methyl-transferase enzyme, CELLSCRIPT, INC.) according toinformation and protocols provided in the product literature.

The present researchers previously found that cap1 mRNA is oftenexpressed into protein at higher levels than the corresponding cap0 mRNAwhen introduced into living cells in culture. Therefore, the use of mRNAthat has a cap1 structure is preferred for all of the methods herein.However, although mRNA that has a cap1 structure is preferred, in someembodiments, mRNA used in the methods has a cap with a cap0 structure,meaning that the penultimate nucleotide with respect to the capnucleotide does not have a methyl group on the 2′-position of theribose. With some but not all transcripts, transfection of eukaryoticcells with mRNA having a cap with a cap1 structure results in a higherlevel or longer duration of protein expression in the transfected cellscompared to transfection of the same cells with the same mRNA but with acap having a cap0 structure. In some embodiments, the mRNA used in themethods of the present invention has a modified cap nucleotide.

In some experiments performed prior to the experiments presented in theEXAMPLES herein, the present Applicants found that, when 1079 or IMR90human fibroblast cells were transfected with OCT4 mRNA that containedeither uridine or pseudouridine in place of uridine, thepseudouridine-containing mRNA was expressed at a higher level or for alonger duration than the mRNA that contained uridine. Therefore, in somepreferred embodiments, one or more or all of the uridines contained inthe mRNA(s) used in the methods of the present invention is/are replacedby pseudouridine (e.g., by substituting pseudouridine-5′-triphosphate inthe IVT reaction to synthesize the RNA in place ofuridine-5′-triphosphate). However, in some embodiments, the mRNA used inthe methods of the invention contains uridine and does not containpseudouridine. In addition, in order to accomplish specific goals, anucleic acid base, sugar moiety, or internucleoside linkage in one ormore of the nucleotides of the ssRNA or mRNA that is introduced into aeukaryotic cell in the methods of the invention may comprise a modifiednucleic acid base, sugar moiety, or internucleoside linkage.

The invention is also not limited with respect to the source of the invitro-synthesized ssRNA or mRNA that is delivered into the eukaryoticcell in the methods of the invention. In some embodiments, such as thosedescribed in the EXAMPLES, the ssRNA or mRNA is synthesized in vitro bytranscription of a DNA template comprising a gene cloned in a linearizedplasmid vector or a PCR or RT-PCR amplification product, capping using acapping enzyme system, and polyadenylation using a poly-A polymerase. Insome other embodiments, the ssRNA or mRNA that is delivered into theeukaryotic cell is derived from a cell or a biological sample. Forexample, in some embodiments, the mRNA derived from a cell or biologicalsample is obtained by amplifying the mRNA from the cell or biologicalsample using an RNA amplification reaction. In some preferredembodiments, the mRNA derived from the cell or biological sample isamplified to generate sense RNA according to the methods described inU.S. Pat. No. 8,039,214, which is incorporated herein by reference.

With respect to the methods comprising introducing mRNA encoding one ormore iPSC cell induction factors in order to generate a dedifferentiatedcell (e.g., an iPS cell), the invention is not limited by the nature ofthe iPS cell induction factors used. Any mRNA encoding one or moreprotein induction factors now known, or later discovered, that find usein dedifferentiation, are contemplated for use in the present invention.In some embodiments, one or more mRNAs encoding for KLF4, LIN28,wild-type c-MYC, mutant c-MYC(T58A) (Wang X et al., 2011; Wasylishen AR, et al. 2011), L-MYC, NANOG, OCT4, or SOX2 are employed. OCT-3/4proteins and certain protein members of the SOX gene family (SOX1, SOX2,SOX3, and SOX15) have been identified as transcriptional regulatorsinvolved in the induction process. Additional genes encode certainprotein members the KLF family (KLF1, KLF2, KLF4, and KLF5), the MYCfamily (c-MYC(WT), c-MYC(T58A), L-MYC, and N-MYC), NANOG, and LIN28,which have been identified to increase the induction efficiency. One ormore these factors may be used in certain embodiments.

While the compositions and methods of the invention may be used togenerated iPS cells, the invention is not limited to the generation ofsuch cells. For example, in some embodiments, mRNA encoding one or morereprogramming factors is introduced into a cell in order to generate acell with a changed state of differentiation compared to the cell intowhich the mRNA was introduced. For example, in some embodiments, mRNAencoding one or more iPS cell induction factors is used to generate adedifferentiated cell that is not an iPS cell. Such cells find use inresearch, drug screening, and other applications.

In some embodiments, the present invention further provides methodsemploying the dedifferentiated cells generated by the above methods. Forexample, such cells find use in research, drug screening, andtherapeutic applications in humans or other animals. For example, insome embodiments, the cells generated find use in the identification andcharacterization of iPS cell induction factors as well as other factorsassociated with differentiation or dedifferentiation. In someembodiments, the generated dedifferentiated cells are transplanted intoan organism or into a tissue residing in vitro or in vivo. In someembodiments, an organism, tissue, or culture system housing thegenerated cells is exposed to a test compound and the effect of the testcompound on the cells or on the organism, tissue, or culture system isobserved or measured.

In some other embodiments, a dedifferentiated cell generated using theabove methods (e.g., an iPS cell) is further treated to generate adifferentiated cell that has the same state of differentiation or celltype compared to the somatic cell from which the dedifferentiated cellwas generated. In some other embodiments, the dedifferentiated cellgenerated using the above methods (e.g., an iPS cell) is further treatedto generate a differentiated cell that has a different state ofdifferentiation or cell type compared to the somatic cell from which thededifferentiated cell was generated. In some embodiments, thedifferentiated cell is generated from the generated dedifferentiatedcell (e.g., the generated iPS cell) by introducing mRNA encoding one ormore reprogramming factors into the generated iPS cell and maintainingthe cell into which the mRNA is introduced under conditions wherein thecell is viable and is differentiated into a cell that has a changedstate of differentiation or cell type compared to the generateddedifferentiated cell (e.g., the generated iPS cell) into which the mRNAencoding the one or more reprogramming factors is introduced. In some ofthese embodiments, the generated differentiated cell that has thechanged state of differentiation is used for research, drug screening,or therapeutic applications (e.g., in humans or other animals). Forexample, the generated differentiated cells find use in theidentification and characterization of reprogramming factors associatedwith differentiation. In some embodiments, the generated differentiatedcells are transplanted into an organism or into a tissue residing invitro or in vivo.

In some embodiments, an organism, tissue, or culture system housing thegenerated differentiated cells is exposed to a test compound and theeffect of the test compound on the cells or on the organism, tissue, orculture system is observed or measured.

In some preferred embodiments of the method comprising introducing mRNAencoding one or more iPSC induction factors into a somatic cell andmaintaining the cell under conditions wherein the cell is viable and themRNA that is introduced into the cell is expressed in sufficient amountand for sufficient time to generate a dedifferentiated cell (e.g.,wherein the dedifferentiated cell is an induced pluripotent stem cell),the sufficient time to generate a dedifferentiated cell is less than oneweek. However, in some embodiments of the method, the sufficient time togenerate a dedifferentiated cell (e.g., an iPS cell) is at least eightdays. In some embodiments of the method, the sufficient time to generatea dedifferentiated cell (e.g., an iPS cell) is greater than eight days(e.g., 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16days, 17 days, 18 days, or more). Among other factors the particular iPScell induction factors used and their doses and relative doses, as wellas the feeder cells (if used), media and other growth conditions affectthe amount of time that is sufficient time to generate adedifferentiated cell (e.g., an iPS cell). For example, the presentapplicants found that under the same culture conditions, the use ofssRNA molecules encoding L-MYC required a longer time (e.g., at leastabout 17 days) to generate iPS cells than when ssRNA molecules encodingc-MYC were used (e.g., in one experiment, requiring only about 10-12days to generate iPS cells). In some preferred embodiments of thismethod, the reprogramming efficiency for generating dedifferentiatedcells is greater than or equal to 50 dedifferentiated cells (e.g.,iPSCs) per 3×10⁵ input cells into which the mRNA is introduced. In somepreferred embodiments of this method, the reprogramming efficiency forgenerating dedifferentiated cells is greater than or equal to 100dedifferentiated cells (e.g., iPSCs) per 3×10⁵ input cells into whichthe mRNA is introduced. In some preferred embodiments of this method,the reprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 150 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 200 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 300 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 400 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 500 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 600 dedifferentiatedcells per 3×10⁵ input cells (e.g., iPSCs) into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 700 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 800 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 900 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 1000 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. Thus, in some preferred embodiments, this method was greaterthan 2-fold more efficient than the published protocol comprisingdelivery of reprogramming factors with a viral vector (e.g., alentivirus vector). In some preferred embodiments, this method wasgreater than 5-fold more efficient than the published protocolcomprising delivery of reprogramming factors with a viral vector (e.g.,a lentivirus vector). In some preferred embodiments, this method wasgreater than 10-fold more efficient than the published protocolcomprising delivery of reprogramming factors with a viral vector (e.g.,a lentivirus vector). In some preferred embodiments, this method wasgreater than 20-fold more efficient than the published protocolcomprising delivery of reprogramming factors with a viral vector (e.g.,a lentivirus vector). In some preferred embodiments, this method wasgreater than 25-fold more efficient than the published protocolcomprising delivery of reprogramming factors with a viral vector (e.g.,a lentivirus vector). In some preferred embodiments, this method wasgreater than 30-fold more efficient than the published protocolcomprising delivery of reprogramming factors with a viral vector (e.g.,a lentivirus vector). In some preferred embodiments, this method wasgreater than 35-fold more efficient than the published protocolcomprising delivery of reprogramming factors with a viral vector (e.g.,a lentivirus vector). In some preferred embodiments, this method wasgreater than 40-fold more efficient than the published protocolcomprising delivery of reprogramming factors with a viral vector (e.g.,a lentivirus vector).

The present invention further provides compositions (systems, kits,reaction mixtures, cells, mRNA) used or useful in the methods and/orgenerated by the methods described herein. For example, in someembodiments, the present invention provides an mRNA encoding an iPS cellinduction factor, the mRNA having pseudouridine in place of uridine.

The present invention further provides compositions comprising atransfection reagent and an mRNA encoding an iPS cell induction factor(e.g., a mixture of transfection reagent and mRNA).

In some embodiments, the compositions comprise mRNA encoding a plurality(e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 6) of iPS cellinduction factors, including, but not limited to, KLF4, LIN28, c-MYC,NANOG, OCT4, and SOX2.

The compositions may further comprise any other reagent or componentsufficient, necessary, or useful for practicing any of the methodsdescribed herein. Such reagents or components include, but are notlimited to, transfection reagents, culture medium (e.g., MEF-conditionmedium), cells (e.g., somatic cells, iPS cells), containers, boxes,buffers, inhibitors (e.g., RNase inhibitors), labels (e.g., fluorescent,luminescent, radioactive, etc.), positive and/or negative controlmolecules, reagents for generating capped mRNA, dry ice or otherrefrigerants, instructions for use, cell culture equipment,detection/analysis equipment, and the like.

In certain embodiments, the ssRNAs or mRNAs comprising a composition,kit or method of the invention are purified or treated to generatepurified or treated RNA compositions or ssRNA or mRNA preparations thathave most of the contaminating RNA molecules removed (e.g., moleculesthat cause an immunogenic response in the cells). In certainembodiments, the ssRNA or mRNA used in the purified or treated RNAcomposition or preparation is purified to remove the contaminants,including the RNA contaminants (e.g., dsRNA contaminants) so that it issubstantially, practically free, extremely free or absolutely free ofsaid contaminants. The present invention is not limited with respect tothe purification or treatment methods used to purify the ssRNA or mRNAfor methods herein that use a purified or treated RNA composition (e.g.,comprising a ssRNA or mRNA) to induce a biological or biochemical effect(e.g, for reprogramming a human or mammalian cell from a firstdifferentiated state or phenotype to a second differentiated state orphenotype), and the invention includes use of any method that is knownin the art or developed in the future in order to purify the ssRNA ormRNA and remove contaminants, including RNA contaminants, that interferewith the intended use of the ssRNA or mRNA. For example, in preferredembodiments, the purification of the ssRNA or mRNA removes contaminantsthat are toxic to the cells (e.g., by inducing an innate immune responsein the cells, or, in the case of RNA contaminants comprising dsRNA, byinducing RNA interference (RNAi), e.g., via siRNA or long RNAimolecules) and contaminants that directly or indirectly decreasetranslation of the mRNA in the cells). In some embodiments, the ssRNA ormRNA is purified by HPLC using a method described herein, including inthe EXAMPLES. In certain embodiments, the ssRNA or mRNA is purifiedusing on a polymeric resin substrate comprising a C18 derivatizedstyrene-divinylbenzene copolymer and a triethylamine acetate (TEAA) ionpairing agent is used in the column buffer along with the use of anacetonitrile gradient to elute the ssRNA or mRNA and separate it fromthe RNA contaminants in a size-dependent manner; in some embodiments,the ssRNA or mRNA purification is performed using HPLC, but in someother embodiments a gravity flow column is used for the purification. Insome embodiments, the ssRNA or mRNA is purified using a method describedin the book entitled “RNA Purification and Analysis” by Douglas T.Gjerde, Lee Hoang, and David Homby, published by Wiley-VCH, 2009, hereinincorporated by reference. In some embodiments, the ssRNA or mRNApurification is carried out in a non-denaturing mode (e.g., at atemperature less than about 50 degrees C., e.g., at ambienttemperature). In some embodiments, the ssRNA or mRNA purification iscarried out in a partially denaturing mode (e.g., at a temperature lessthan about 50 degrees C. and 72 degrees C.). In some embodiments, thessRNA or mRNA purification is carried out in a denaturing mode (e.g., ata temperature greater than about 72 degrees C.). Of course, those withknowledge in the art will know that the denaturing temperature dependson the melting temperature (Tm) of the ssRNA or mRNA that is beingpurified as well as on the melting temperatures of RNA, DNA, or RNA/DNAhybrids which contaminate the ssRNA or mRNA. In some other embodiments,the ssRNA or mRNA is purified as described by Mellits K H et al., 1990).After observing that incubation of in vitro-transcribed RNA (IVT-RNA)with RNase III using conditions as described by Robertson et al.(Robertson, H D et al., 1968) antagonized activation of DAI in acell-free in vitro translation system, these authors used a three steppurification to remove the contaminants which may be used in embodimentsof the present invention. Step 1 was 8% polyacrylamide gelelectrophoresis in 7 M urea (denaturing conditions). The major RNA bandwas excised from the gel slice and subjected to 8% polyacrylamide gelelectrophoresis under nondenaturing condition (no urea) and the majorband recovered from the gel slice. Further purification was done on acellulose CF-1I column using an ethanol-salt buffer mobile phase whichseparates double stranded RNA from single stranded RNA (Franklin R M.1966. Proc. Natl. Acad. Sci. USA 55: 1504-1511; Barber R. 1966. Biochem.Biophys. Acta 114:422; and Zelcer A et al. 1982. J. Gen. Virol. 59:139-148, all of which are herein incorporated by reference) and thefinal purification step was cellulose chromatography. A similar 3-stepIVT-RNA purification method comprising denaturing gel electrophoresis,non-denaturing gel electrophoresis and CF-11 chromatography was used byPe'ery and Mathews (Pe'ery T and Mathews M B. 1997). These authors saidthat RNase III might be an optional pretreatment or in place of thenondenaturing gel, provided that the RNA was not sensitive to theenzyme, which they observed cut some ssRNAs. In some other embodiments,the ssRNA or mRNA is purified using an hydroxylapatite (HAP) columnunder either non-denaturing conditions or at higher temperatures (e.g.,as described by Pays E. 1977. Biochem. J. 165: 237-245; Lewandowski L Jet al. 1971. J. Virol. 8: 809-812; Clawson G A and Smuckler E A. 1982.Cancer Research 42: 3228-3231; and/or Andrews-Pfannkoch C et al. 2010.Applied and Environmental Microbiology 76: 5039-5045, all of which areherein incorporated by reference). In some other embodiments, the ssRNAor mRNA is purified by weak anion exchange liquid chromatography undernon-denaturing conditions (e.g., as described by Easton L E et al. 2010.RNA 16: 647-653 to clean up in vitro transcription reactions, hereinincorporated by reference). In some embodiments, the ssRNA or mRNA ispurified using one or more of any of the methods described herein or anyother method known in the art or developed in the future. In stillanother embodiment, the ssRNA or mRNA used in the compositions, kits ormethods of the present invention is purified using a process whichcomprises treating the ssRNA or mRNA with an enzyme that specificallyacts (e.g., digests) one or more contaminant RNA or contaminant nucleicacids (e.g., including DNA), but which does not act on (e.g., does notdigest) the desired ssRNA or mRNA. For example, in some embodiments, thessRNA or mRNA used in the compositions and methods of the presentinvention is purified using a process which comprises treating the mRNAwith a ribonuclease III (RNase III) enzyme (e.g., E. coli RNase III) andthe ssRNA or mRNA is then purified away from the RNase III digestionproducts. A ribonuclease III (RNase III) enzyme herein means an enzymethat digests dsRNA greater than about twelve basepairs to short dsRNAfragments. In some embodiments, the ssRNA or mRNA used in thecompositions, kits or methods of the present invention is purified usinga process which comprises treating the ssRNA or mRNA with one or moreother enzymes that specifically digest one or more contaminant RNAs orcontaminant nucleic acids (e.g., including DNA).

In some embodiments, the results described herein demonstrate a methodof the present invention for reprogramming a cell that exhibits a firststate of differentiation or phenotype to a cell that exhibits a secondstate of differentiation or phenotype(e.g., reprogramming a mousemesenchymal stem cell to a myoblast cell; e.g., reprogramming a humanfibroblast cell to a neuron cell; ore.g., reprogramming a somatic cell;e.g., a fibroblast, keratinacyte or blood cell to a dedifferentiated oriPS cell), comprising: repeatedly (e.g., on or during each of 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18 or >18 days) orcontinuously introducing into a cell that exhibits a first state ofdifferentiation or phenotype an mRNA reprogramming mix comprisingpseudouridine-modified (GAΨC) mRNA encoding one or more proteinreprogramming factors (e.g., one or more transcription factors), whereinthe GAΨC mRNA: (i) exhibits a cap on its 5′ terminus and a polyA tail onits 3′ terminus; (ii) is purified (e.g., by HPLC or gravity-flow orlow-pressure chromatography or electrophoresis) or treated with adsRNA-specific endoribonuclease (e.g., RNase III) under conditionswherein: e.g., for mRNA encoding MYOD protein, less than 1% of the totalRNA comprising said mRNA reprogramming mix used for said introducingcomprises dsRNA; e.g., for mRNA encoding protein reprogramming factorsfor inducing neuron cells or iPS cells, less than 0.01% of the total RNAcomprising said mRNA reprogramming mix used for said introducingcomprises dsRNA; and maintaining the cell under conditions to generate areprogrammed cell that exhibits a second state of differentiation orphenotype. In some preferred embodiments, the cap exhibits a cap1structure, wherein the 5′ penultimate nucleotide comprises a 2′-O-methylgroup.

In certain other embodiments, these results demonstrate a method of thepresent invention for reprogramming a cell that exhibits a first stateof differentiation (e.g., a somatic cell; e.g., a fibroblast,keratinacyte, a blood cell) or phenotype to a cell that exhibits asecond state of differentiation (e.g., a dedifferentiated or iPS cell),comprising: repeatedly (e.g., on or during each of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, or >20 days) orcontinuously introducing into the cell that exhibits a first state of orphenotype an mRNA reprogramming mix comprising unmodified GAUC mRNAencoding one or more protein reprogramming factors (e.g., one or moretranscription factors), wherein the unmodified GAUC mRNA: (i) exhibits acap on its 5′ terminus and a polyA tail on its 3′ terminus; (ii) ispurified (e.g., by HPLC or gravity-flow or low-pressure chromatograph orelectrophoresis) or treated with a dsRNA-specific endoribonuclease(e.g., RNase III) under conditions wherein (e.g., for mRNA encoding MYODprotein, less than 0.1% of the total RNA comprising said mRNAreprogramming mix used for said introducing comprises dsRNA; e.g., formRNA encoding protein reprogramming factors for inducing iPS cells, lessthan 0.004% of the total RNA comprising said mRNA reprogramming mix usedfor said introducing comprises dsRNA); And culturing the cell underconditions to generate a reprogrammed cell that exhibits a second stateof differentiation or phenotype. In some preferred embodiments, the capexhibits a cap1 structure, wherein the 5′ penultimate nucleotidecomprises a 2′-O-methyl group. In some preferred embodiments of themethod for reprogramming a cell that exhibits a first differentiatedstate or phenotype to a cell that exhibits a second differentiated stateor phenotype, the cell that exhibits a first differentiated state orphenotype is a somatic cell (e.g., a human fibroblast or keratinocytecell), the mRNA reprogramming mix used for said introducing (e.g.,transfection) into the cell that exhibits the first state ofdifferentiation comprises either pseudouridine-modified GAΨC mRNAs,pseudouridine- and 5-methylcytidine-modified GAΨm⁵C mRNAs, or unmodifiedGAUC mRNAs that exhibit a cap1 structure, in each case with polyA-tailswith at least 50 A nucleosides (e.g., about 150 A nucleosides); whereinsaid mRNA reprogramming mix encodes a mix selected from among:K₍₁₋₃₎MO₃S; K₍₁₋₃₎MO₃SL; and K₍₁₋₃₎MO₃SLN; wherein M=c-MYC(T58A) orc-MYC; and wherein said the mRNAs in said mRNA reprogramming mix areRNase III-treated and are absolutely free of dsRNA; and the cell thatexhibits a second differentiated state or phenotype is an or iPS cell.

EXAMPLES

The present invention will now be illustrated by the following examples,which are not to be considered limiting in any way.

General Materials and Methods, Particularly Those Pertaining toDevelopment of the RNase III Treatment Method.

The following description illustrates examples of the materials andmethods generally used herein. Whenever possible, applicants have triedto point out when other materials or methods, or deviations from ormodifications of the general materials and methods were used inparticular EXAMPLES or experiments described below. However, those withknowledge, after reading the descriptions below, will understand howmodify the specific embodiments described without deviating from thescope of the present invention.

Production of an RNA Substrate Comprising Both dsRNA and ssRNA Portionsfor Use in Simultaneously Assaying RNase III Activities on Both dsRNAand ssRNA.

A T7 and T3 RNA polymerase promoter-containing plasmid DNA construct wasused for generation of an RNA substrate comprising a dsRNA centralportion with a 5′-terminal ssRNA portion on one strand and a 3′-terminalssRNA portion on the other strand for use in assaying RNase IIIactivities on both dsRNA and ssRNA simultaneously. A 1671-basepairinsert, as shown in FIG. 1, was cut from the plasmid backbone with ClaIand then single-stranded RNA (ssRNA) was generated by in vitrotranscription of each DNA strand (FIG. 1) in two separate reactionsusing either a T7-Scribe™ Standard RNA IVT Kit (CELLSCRIPT, INC.,Madison, Wis., USA) or an AmpliScribe™ T3 High Yield transcription Kit(epicentre, Madison, Wis.), respectively. Following DNase I treatment toremove the DNA template, the ssRNA transcripts were precipitated withone volume of 5 M ammonium acetate and were resuspended in 10 mMTris-HCl (pH 7.5) with 1 mM EDTA. The two strands of ssRNA were annealedby incubating equivalent amounts of the T7- and T3-transcribed ssRNAs at94° C. for 2 minutes, 72° C. for 10 minutes and then slowly cooling toroom temperature. The resulting annealed RNA was 1671 bases in lengthwith a 255-base single-stranded region on one end and a 136-basesingle-stranded region on the other end.

Production of a Control ssRNA

A T7 RNA polymerase promoter-containing plasmid construct with a 955base insert was linearized with EcoRI. The T7-Scribe™ Standard RNA IVTKit was used to transcribe RNA from the template. DNase I treatment andammonium acetate precipitation were performed as described above and thessRNA transcript was resuspended in water.

Simultaneous Assay of RNase III Activity on dsRNA and ssRNA SubstratesUnder Different Reaction Conditions

One microgram of the RNA substrate comprising both dsRNA and ssRNAportions, (referred to herein as either the “RNA substrate” or the“dsRNA substrate”) was adjusted to a final concentration of 20ng/microliter, and treated with 20 nM RNase III using the incubatedMINiMMUNE™ dsRNA removal kit (CELLSCRIPT, INC., Madison, Wis., USA) at37° C. for 10 minutes in a 50-microliter reaction mixture that varied incomposition. In one embodiment, the reaction mixture contained finalconcentrations of 33 mM Tris-acetate (pH 8) as a buffer, 200 mMpotassium acetate as a monovalent salt, and between 1 mM to 10 mMmagnesium acetate as the divalent magnesium cation source. Reactionsalso contained 0.8 units per microliter SCRIPTGUARD™ RNase inhibitor(CELLSCRIPT, INC., Madison, Wis., USA). The reactions were stopped bythe addition of EDTA to the same final concentration as theconcentration of divalent magnesium cations used in the assay (e.g., 1mM to 10 mM final).

Digestion of the RNA substrate was analyzed by denaturing gelelectrophoresis. Briefly, 10-microliter samples of each 50-microliterRNase III reaction was analyzed by denaturing gel electrophoresis on a1% agarose, 1 M urea gel in 1× TBE buffer. Samples were denatured for 2minutes at 94° C. in formamide-containing loading buffer and run next toRNA Millennium™ markers (Ambion/Life Technologies). Gels were stainedwith SYBR® Gold nucleic acid gel stain (Invitrogen/Life Technologies).

Dot Blot Assays for Assay or Quantification of dsRNA UsingdsRNA-Specific Antibodies

Appropriate dilutions of RNA samples (5 microliters/sample) for theintended assay purpose were applied to Nytran SPC positively chargednylon membranes (Thermo Scientific, Waltham, Mass.). The RNA was allowedto dry on the nylon membrane for 30 minutes at room temperature. Themembranes were then blocked in blocking buffer (25 mM Tris pH 7.5, 150mM NaCl, 0.05% Tween 20, 5% W/V dry milk) at room temperature for 1 houron a rotating platform. The primary antibodies (J2 or K1 antibodies,English & Scientific Consulting, Szirák, Hungary) were then added at 1microgram/ml in blocking buffer at room temperature for 1 hour on arotating platform. The membranes were then washed 6 times for 5 minuteswith 20 mls of wash buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05%Tween 20). Membranes were then incubated at room temperature for 1 houron a rotating platform in blocking buffer to which a 1:1000 dilution ofthe secondary antibody (Anti-mouse IgG HRP, Cell Signaling Technologies,Danvers, Mass.) was added. The membranes were again washed 6 times for 5minutes with 20 mls of wash buffer (25 mM Tris pH 7.5, 150 mM NaCl,0.05% Tween 20). Then, equal volumes of SUPERSIGNAL WEST PICO™Chemiluminescent Substrates (Thermo Scientific, Waltham, Mass.) wereadded and the color was allowed to develop for 5 minutes on a rotatingplatform. The dots were imaged by exposing the film in the dark room andthen developing the film in Kodak Developer (Sigma, St. Louis, Mo.) for1 minute and Kodak Fixer (Sigma, St. Louis, Mo.) for 1 minute.

General Materials and Methods, Particularly Those Pertaining toReprogramming of Cells Exhibiting a First Differentiated State orPhenotype to a Second Differentiated State or Phenotype (e.g.,Reprogramming of Human Somatic Cells to iPS Cells)

Methods for Using Feeder Cells and Plating BJ Fibroblasts forReprogramming to iPSCs with mRNAs Encoding iPSC Reprogramming Factors.

Nuff cells (Human Foreskin Fibroblast P9 irradiated (donor 11)(GlobalStem, Rockville, Md.) were plated at a density of 5×10⁵cells/well in a gelatin-coated 6-well dish. The Nuffs were grownovernight at 37° C., 5% CO₂ in Nuff culture medium (DMEM Invitrogen cat#11965-118, 10% Hyclone FBS Fisher cat #SH30070.03HI, GLUTAMAX™Invitrogen cat #35050-061, Pen/Strep Invitrogen cat #15140-122). BJFibroblasts (ATCC) were plated at 1×10⁴ cells per well on Nuffs cellswhich had been plated the previous day. The cells were then incubated inBJ Fibroblast medium (Advanced MEM, 10% Hyclone FBS Fisher cat#SH30070.03HI, GLUTAMAX Invitrogen cat #35050-061, Pen/Strep Invitrogencat #15140-122) overnight at 37° C., 5% CO₂.

TransIT™ mRNA Transfection Protocol

BJ fibroblast medium was removed from BJ fibroblasts plated on Nufffeeder cells and replaced by PLURITON™ mRNA reprogramming medium(Stemgent, Cambridge, Mass.)(base medium with supplement andpenicillin/streptomycin) (2 mls) with or without 4 microliters of B18Rrecombinant protein (EBiosciences, San Diego, Calif.) to a finalconcentration of 200 ng/ml. The media were changed immediately beforeeach transfection with Mirus mRNA Transfection Reagent (Mirus Bio,Madison, Wis.). To transfect the BJ fibroblasts, 0.6 to 1.4 microgramsof the 3:1:1:1:1 mRNA mix comprising OCT4, SOX2, KLF4, LIN28 and eitherc-MYC(T58A) or cMYC was added to 120 microliters of OptiMEM medium andthen TransIT™ Boost (2 microliters per microgram of mRNA) and TransIT™mRNA transfection reagent (microliters per microgram of mRNA) (MirusBio) were mixed with the mRNA. The mRNA-TransIT mix was incubated for 2minutes and then added to each well of BJ fibroblasts on Nuff feeders inPLURITON medium. The following day, the PLURITON media were changedbefore transfecting the same dose of mRNA using TransIT Boost andTransIT mRNA transfection reagent. Nuff-conditioned PLURITON medium wasreplaced by PLURITON medium on the sixth day of transfections. Unlessotherwise indicated, a total of 18 transfections were performed for eachreprogramming experiment.

Embryoid Body Spontaneous Differentiation Protocol

Some iPS colonies that were picked and passaged multiple times (at least5 times) were processed for embryoid body spontaneous differentiation aspreviously described (Huangfu et al., 2008). Briefly, the iPS colonieswere dissociated with Collagenase IV and incubated for 8 days inlow-binding 6-well dishes in iPSC medium (DMEM/F12 medium supplementedwith 20% KNOCKOUT™ serum replacer, 0.1 mM L-glutamine, non-essentialamino acids, and penicillin/streptomycin (all from Invitrogen). One halfof the medium was changed every day during the 8 day period. After eightdays in suspension culture, the embryoid bodies were transferred togelatin-coated 6-well dishes in the same medium (DMEM/F12 mediumsupplemented with 20% KNOCKOUT™ serum replacer, 0.1 mM L-glutamine,non-essential amino acids, and penicillin/streptomycin) and incubatedfor an additional 8 days. The cultures were washed in PBS and then fixedin 4% paraformaldehyde in PBS for 30 minutes are room temperature. Thecells were then stained using antibodies that recognize Desmin (ThermoScientific, Fremont, Calif.), a-Smooth muscle actin (SMA) (Sigma, St.Louis Mo.), Alpha fetoprotein (AFP) (Sigma, St. Louis Mo.), SOX17 (R&DSystems, Minneapolis, Minn.), and Class III beta-tubulin (Covance,Emeryville, Calif.). These primary antibodies were recognized with thesecondary antibody anti-mouse 555 fluorescent antibody (Cell SignalingTechnologies, Danvers, Mass.). Images were taken on a TS100epifluorescent Nikon Microscope.

Alkaline Phosphatase Staining as an iPS Cell Marker

Cells are washed once in 1×PBS, followed by fixing in 4%paraformaldehyde in PBS for 5 minutes. The cell were then washed twotimes in PBS followed by two washes in TBST (25 mM Tris, pH 7.5, 150 mMNaCl, 0.5% TWEEN™ 20). The cells were then washed in AP Buffer (0.1 MTris, pH 9.5, 0.1 M NaCl, 5 mM MgCl₂). Then 132 microliters of 50 mg/mlnitroblue tetrazolium (NBT) in 70% dimethylformamide (DMF) and 64microliters of 50 mg/ml bromochloro-indolyl phosphate (BCIP) in 100%dimethylformamide (DMF) were added to each 20 mls of AP buffer, whichwas then added to the cells for 5-10 minutes until stain developed. Oncethe purple color developed, the cells were washed at least three timeswith TBST, and optionally two times with 1×PBS, and stained colonieswere counted, or stored in PBS for imaging and colony counting.

Live Cell Immunostaining of iPSC Colonies with Tra-1-60

TRA-1-60 is considered to be one relatively stringent marker of fullyreprogrammed iPS cells (Chan et al. 2009). The Tra-1-60 live cellimaging was done with the StainAlive Dylight™ 488 Mouse anti-HumanTra-1-60 antibody (Stemgent) according to the manufacturer'sspecifications. Briefly, a sterile, TRA-1-60 antibody (StainAlive™DyLight™ 488 anti-human TRA-1-60 antibody; Stemgent) was diluted 1:100in reprogramming medium. On day 18 of the reprogramming protocol, themedium was removed and the cells were incubated in TRA-1-60-containingmedia for 30 minutes at 37° C. with 5% CO₂. The cells were then washedtwice with medium to remove the unbound antibody and the cells weremaintained in fresh reprogramming medium during immunofluorescentimaging. This antibody allows live cell staining, instead of fixing thecells and sacrificing them for the imaging.

Methods for Fixed Cell Immunostaining of iPSCs

iPSC colonies were washed twice in 1× phosphate-buffered solution (PBS)and fixed in 4% paraformaldehyde in PBS at room temperature for half anhour. After 3 washes in 1×PBS, cells were washed 3 times in wash buffer,(PBS with 0.1% Triton-X100), and blocked for one hour at roomtemperature in blocking solution, 0.1% triton-X100, 1% BSA, 2% FBS inPBS. Primary antibodies were diluted 1:500 in blocking solution andapplied to cells overnight at 4° C. Cells were washed 6 times in washbuffer. Secondary antibodies were diluted 1:1,000 in blocking buffer,were applied for 2 hours at room temperature in the dark. After 6 washeswith wash buffer, cells were washed twice in 1×PBS before imaging.Primary antibodies used were:

OCT4 Rabbit Antibody (Santa Cruz Biotechnology); TRA-1-60 Mouse Antibody(Cell Signaling Technology); LIN28 Mouse Antibody (Cell SignalingTechnology); NANOG Rabbit Antibody (Cell Signaling Technology); SSEA4Mouse Antibody (Cell Signaling Technology); TRA-1-81 Mouse Antibody(Cell Signaling Technology); and DNMT 3B Rabbit Antibody (Cell SignalingTechnology). Secondary antibodies used were: Alexa Fluor® 488Anti-Rabbit (Molecular Probes, Life Technologies) and Alexa Fluor® 555Anti-Mouse (Molecular Probes, Life Technologies).

Construction of DNA Templates for In Vitro Transcription of ssRNAs ormRNAs Encoding iPSC Reprogramming Factors (e.g., iPSC Reprogramming orInduction Factors).

Open reading frames (ORFs) of human most human genes (e.g., KLF4, LIN28,NANOG, OCT4. SOX2) were PCR-amplified from cDNA clones (e.g., OpenBiosystems, Huntsville, Ala.), or, in some cases, the ORF of certaingenes were obtained by RT-PCR from cell total RNA (e.g., c-MYC ORF wasobtained by RT-PCR from HeLa cell total RNA), cloned into a pUC-basedplasmid vector downstream of a T7 RNA polymerase promoter (Mackie 1988,Studier and Moffatt 1986), and sequenced to confirm the accuracy of thecloned ORF. In some preferred embodiments, the above ORFs were ligatedinto EcoRV (for c-MYC) or EcoRV/SpeI (for KLF4, LIN28, NANOG, OCT4, andSOX2) sites between the 5′ and 3′ Xenopus laevis beta-globinuntranslated regions described (Krieg and Melton 1984).

In some specific embodiments, the pUC19-based vector was modified byinserting a T7 promoter followed by the 5′ UTR of Xenopus laevisβ-globin, a multiple cloning site consisting of restriction sites BglII,EcoRV and SpeI for insertion of a gene of interest, and finally the 3′UTR of Xenopus laevis β-globin. Plasmids were linearized with SalI priorto in vitro transcription; for example the T7 RNA polymerase promoter(underlined/bold), 5′ and 3′ Xenopus laevis beta-globin UTRs(underlined/italics), and the SalI restriction site (GTCGAC/underlined)are depicted in SEQ ID NO. 1. The pUC19-based DNA plasmids comprisingSEQ ID NO. 1 with DNA inserts encoding an iPSC induction factor [e.g.,OCT4 (SEQ ID NO. 2), SOX2 (SEQ ID NO. 3), KLF4 (SEQ ID NO. 4), LIN28(SEQ ID NO. 5), NANOG (SEQ ID NO. 6) and MYC; e.g., either cMYCwild-type long (SEQ ID NO. 7), cMYC(T58A) short (SEQ ID NO. 8), cMYCwild-type short (SEQ ID NO. 9), or L-MYC (SEQ ID NO. 10) mRNA] were eachlinearized by overnight incubation with SalI, and then purified byphenol/chloroform or phenol/chloroform/isoamyl alcohol extraction. Thelinear DNA was precipitated with sodium acetate/ethanol precipitationfollowed by a 70% ethanol wash. Linear DNA was reconstituted in waterand run on an agarose gel to check that the plasmid was fullylinearized. The SalI-treated plasmid DNAs were reconstituted in waterand run on an agarose gel to check that all of each plasmid waslinearized for use in in vitro transcription. Then the linearizedplasmid was used as a template for in vitro transcription as describedherein.

In Vitro Synthesis of mRNAs Encoding iPSC Induction Factors forReprogramming

The T7 mSCRIPT™ mRNA production system (CELLSCRIPT, INC, Madison, Wis.,USA) was used to produce unmodified mRNA with a 5′ Cap1 structure and a3′ poly(A) tail (e.g., with approximately 150 A residues). The T7mSCRIPT™ mRNA production system was also used to produce pseudouridine-and or 5-methylcytidine-modified mRNA with a 5′ cap 1 structure and a 3′poly(A) tail (e.g., with approximately 150 A residues), except thatpseudouridine-5′-triphosphate (TRILINK, San Diego, Calif. or CELLSCRIPT,INC.) or 5-methylcytidine-5′-triphosphate (TRILINK, San Diego, Calif.)was used in place of uridine-5′-triphosphate orcytidine-5′-triphosphate, respectively, in the in vitro transcriptionreactions. For example, the linearized templates were used for in vitrotranscription as described in the literature provided with the T7mSCRIPT™ standard mRNA production system (CELLSCRIPT, INC., Madison,Wis., USA), as follows: 1 microgram of linear DNA template was used in1× reactions along with 2 microliters of 10× T7-SCRIBE™ transcriptionbuffer, 1.8 microliters of 100 mM ATP, 1.8 microliters of 100 mM CTP orm⁵CTP (Trilink Biotechnologies, San Diego, Calif.), 1.8 microliters of100 mM GTP, 1.8 microliters of 100 mM UTP or 4P (CELLSCRIPT), 2microliters of 100 mM DTT, 0.5 microliter SCRIPTGUARD™ RNase inhibitor,and 2 microliters T7-SCRIBE™ enzyme solution.

Following in vitro transcription (IVT), the DNA templates were digestedwith DNase I and then in vitro-transcribed mRNAs were cleaned up byphenol-chloroform extraction and ammonium acetate precipitation asdescribed in the RNA Quick Cleanup Method section. Briefly, the in vitrotranscription reactions were incubated at 37° C. for 1 hour followed byadding 1 microliter of RNase-free DNase I and incubating for 15additional minutes at 37° C. The RNA is precipitated by adding an equalvolume of 5M ammonium acetate followed by incubation on ice for 10minutes. Then the RNA is pelleted by spinning at 13,000 rpms for 10minutes. The pellet is washed with 70% ethanol and resuspended in water.

The in vitro-transcribed mRNAs were then treated with RNase III asdescribed in the RNase III treatment method section, after which themRNAs were again cleaned up as described in the RNA Quick Cleanup Methodsection.

Each of the mRNAs was then capped to cap1 mRNA (or in other embodiments,to cap0 mRNA) using the SCRIPTCAP™ capping enzyme and the SCRIPTCAP™2′-O-methyl-transferase enzymes (or for cap0 mRNA, only the SCRIPTCAP™capping enzyme) as described in the T7 mSCRIPT™ standard mRNA productionsystem: Briefly, 60 micrograms of in vitro-transcribed RNA was added to10 microliters of SCRIPTCAP™ capping buffer, 5 microliters of 20 mM GTP,2.5 microliters of S-adenosyl-methionine (SAM), 2.5 microliters ofSCRIPTGUARD™ RNase Inhibitor, 4 microliters of SCRIPTCAP™2′-O-Methyltransferase, 4 microliters of SCRIPTCAP™ capping enzyme, andwater to 100 microliters. All capping reactions were incubated at 37° C.for 1 hour followed by going directly into the poly(A) tailing reaction.

Synthesis of Poly (A) Tailed mRNA was performed using the T7 m SCRIPT™RNA production system (CELLSCRIPT, Inc.) as follows: 12 microliters of10× A-Plus Tailing Buffer, 6 microliters of 20 mM ATP, 5 microliters ofA-PLUS™ poly(A) polymerase, and 0.5 microliter of SCRIPTGUARD™ RNaseinhibitor were added to the 100 microliters of 5′-capped invitro-transcribed RNA and incubated at 37° C. for 30 minutes (togenerate a poly(A) tail of approximately 150 bases) or for 1 hour (togenerate a poly(A) tail of >200 bases. Capped and polyadenylated mRNAswere cleaned up as described in the RNA Quick Cleanup Method section oras otherwise described in the T7 mSCRIPT™ standard mRNA productionsystem. Thus, reactions were terminated by two phenol/chloroform/isoamylextractions followed by precipitation with an equal volume of 5Mammonium acetate. The mRNA/5M ammonium acetate mixes were spun at 13,000rpm for 10 minutes, washed in 70% ethanol and resuspended in sterilewater.

In some experiments, the in vitro-transcribed mRNAs encoding iPSCreprogramming factors were evaluated for expression followingtransfection of human cells. For example, in some experiments, invitro-transcribed cap1, poly(A)-tailed (with ˜150 A nucleotides) mRNAsmade with pseudouridine-5′-triphosphate substituting foruridine-5′-triphosphate (Kariko et al., 2008) and encoding KLF4, LIN28,c-MYC, NANOG, OCT4 or SOX2 each resulted in expression and propersubcellular localization of each respective protein product in newbornfetal foreskin 1079 human fibroblasts. For example, in some experiments,1079 fibroblasts were transfected with up to 4 micrograms of one ofthese mRNAs per well of a 6-well dish and then analyzed byimmunofluorescence analysis 24 hours post-transfection. Briefly, the1079 cells were washed with PBS and fixed in 4% paraformaldehyde in PBSfor 30 minutes at room temperature, then washed 3 times for 5 minuteseach wash with PBS followed by three washes in PBS+0.1% Triton X-100,blocked in blocking buffer (PBS+0.1% Triton, 2% FBS, and 1% BSA) for 1hour at room temperature, and then incubated for 2 hours at roomtemperature with the primary antibody (e.g., mouse anti-human OCT4 Cat#sc-5279, Santa Cruz Biotechnology, Santa Cruz, Calif.; rabbitanti-human NANOG Cat #3580; rabbit anti-human KLF4 Cat #4038; mouseanti-human LIN28 Cat #5930; rabbit anti-human c-MYC Cat #5605; or rabbitanti-human SOX2 Cat #3579; all from Cell Signaling Technology, Beverly,Mass.) at a 1:500 dilution in blocking buffer. After washing 5 times inPBS+0.1% Triton X-100, the cells were incubated for 2 hours withanti-rabbit ALEXA Fluor 488 antibody (Cat #4412, Cell SignalingTechnology), anti-mouse FITC secondary (Cat #F5262, Sigma), or ananti-mouse Alexa Fluor 555 (Cat #4409, Cell Signaling Technology) at1:1000 dilutions in blocking buffer. Images were taken on a Nikon TS100Finverted microscope (Nikon, Tokyo, Japan) with a 2-megapixel monochromedigital camera (Nikon) using NIS-elements software (Nikon). EndogenousKLF4, LIN28, NANOG, OCT4 and SOX2 protein levels were undetectable byimmunofluorescence in untransfected 1079 cells, although, in some cases,endogenous levels of c-MYC were relatively high in untransfected 1079cells. Transfections with mRNAs encoding the transcription factors,KLF4, c-MYC, NANOG, OCT4, and SOX2 all resulted in primarily nuclearlocalization of each protein 24 hours after mRNA transfections, whereasthe cytoplasmic mRNA binding protein, LIN28, was localized to thecytoplasm.

Example of Use of the In Vitro-Transcribed Capped and Poly(A)-TailedmRNAs Encoding iPSC Reprogramming Factors for Reprogramming Human orMouse Somatic Cells to iPS Cells

Unless otherwise indicated for a particular experiment, the mRNAreprogramming factors used in methods for induction of iPSCs werediluted to 100 ng/ml and a mix was made containing the factors in a3:1:1:1:1 molar ratio of OCT4/SOX2/KLF4/LIN28/MYC (e.g. cMYC, cMYC(T58A)or L-MYC), and aliquoted into aliquots containing about 1 to 1.4micrograms of total RNA. For example, in one embodiment comprising useof 1.1 micrograms total per day per well of mRNA for reprogramming, wasprepared in a 3:1:1:1:1 molar ratio of OCT4, SOX2, KLF4, LIN28 and c-MYCor c-MYC(T58A) by mixing the following volumes of a 100 ng/ml solutionof each mRNA reprogramming factor: OCT4, 385.1 microliters; SOX2, 119.2microliters; KLF4, 155.9 microliters; LIN28, 82.5 microliters; c-MYC orc-MYC(T58A), 147.7 microliters; plus 109.6 microliters of water, makinga total volume of 1 ml. [Alternatively, in some embodiments, a portionof the water was replaced by an aqueous solution of mRNA encodingenhanced green fluorescent protein (EGFP) at 100 ng/ml as a transfectionmarker.]

RNA Quick Cleanup Method

The protocol below provides quick RNA cleanup method for removal ofenzymes, nucleotides, small oligonucleotides, and other in vitrotranscription (IVT) or RNase III treatment reaction components from RNA.It is not intended as a method for extensive purification of ssRNA ormRNA. This cleanup method comprises phenol-chloroform extractionfollowed by ammonium acetate precipitation to removes protein andselectively precipitate RNA, leaving residual undigested DNA andunincorporated nucleoside-5′-triphosphates in the supernatant. Withoutlimiting the method with respect to specific RNA quantities purified orspecific volumes of reagents, which can be scaled or adjusted, oneembodiment of the method used with respect to the present invention ispresented below.

-   -   1. Adjust a 20-microliter IVT reaction volume to 200 microliters        total using RNase-Free Water (add 179 microliters to the        reaction).    -   2. Add one volume (200 microliters) of TE-saturated        phenol/chloroform. Vortex for 10 seconds.    -   3. Spin in a microcentrifuge at >10,000×g for 5 minutes to        separate the phases.    -   4. Remove the aqueous (upper) phase with a pipette and transfer        to a clean tube.    -   5. Add one volume (200 microliters) of 5 M ammonium acetate, mix        well then incubate for 15 minutes on ice.    -   6. Pellet the RNA by centrifugation at >10,000×g for 15 minutes        at 4 degrees C.    -   7. Remove the supernatant with a pipette and gently rinse the        pellet with 70% ethanol.    -   8. Remove the 70% ethanol with a pipette without disturbing the        RNA pellet.    -   9. Allow the pellet to dry, then resuspend in 50-75 microliters        of RNase-free water and quantify the RNA by spectrophotometry or        fluorimetry.

Example of an RNase III Treatment Method of the Present Invention

One hundred micrograms of in vitro-transcribed ssRNA, capped and/orpolyadenylated ssRNA, or mRNA, which has preferably been cleaned upusing the RNA Quick Cleanup Method described herein, is incubated in a200-microliter reaction mixture containing 33 mM Tris-acetate (pH 8) asa buffer, 200 mM potassium acetate as a monovalent salt, and betweenabout 1 mM and 4 mM (more preferably, about 2-3 mM, and most preferably2 mM) magnesium acetate as the magnesium salt, and 20 nM RNAse III(CELLSCRIPT, INC., Madison, Wis. 53713) for 30 minutes at 37° C. Unlessotherwise stated the ssRNAs were treated with RNase III using the RNaseIII treatment described herein with 1 or 2 mM magnesium acetate and 150mM potassium acetate. However, in some experiments described herein, upto about 10 mM magnesium acetate was used for the RNase III treatment inorder to evaluate the effect of different divalent magnesium cationconcentrations on the activity of RNase III for dsRNA removal. (In someembodiments, the RNAse III treatment reactions also contain an RNaseinhibitor (e.g., 0.8 units/microliter SCRIPTGUARD™ RNase inhibitor;CELLSCRIPT, INC.). The RNase III treatment reactions are stopped by theaddition of EDTA to a concentration sufficient to complex the magnesiumcations (e.g., 1 mM EDTA final if 1 mM magnesium acetate is used). Inpreferred embodiments, the ssRNA or mRNA is further cleaned up using theRNA Quick Cleanup method, which comprises extraction using TE-saturatedphenol/chloroform, precipitation with 1 volume of 5 M ammonium acetate,and washing of the RNA pellet with 70% ethanol (as described herein forthe RNA Quick Cleanup Method). In some embodiments, the RNaseIII-treated ssRNA or mRNA is then resuspended in water.

Example 1: Magnesium Cation Concentration During RNase III Treatment hasImportant Effects on ssRNA Integrity and the Completeness of RNase IIIDigestion of dsRNA

One microgram of dsRNA was treated with 20 nanomolar RNase III inreaction buffers containing from 0 to 10 mM magnesium acetate in thebuffer. The ideal treatment conditions would digest the 1671-nucleotidelong dsRNA region of the transcript and leave two single-stranded RNAfragments of 255 and 136 nucleotides in length intact (FIG. 1).

As shown in FIG. 2, the dsRNA band was digested by the RNase III. Mostimportantly, the ssRNA bands were of the correct size and intact, basedon minimal smearing below the bands, at magnesium acetate concentrationsbetween about 1 and 4 mM. The fact that the amount of smearing below thessRNA bands steadily increased, beginning at about 5 mM and steadilybecoming worse as magnesium acetate concentrations increased to 10 mM,indicated that an optimal concentration of magnesium acetate for RNaseIII digestion was in the range of about 1 mM to about 4 mM, and morepreferably, about 1 mM to 3 mM. This was a big surprise, because thosewho had worked in the art on RNase III, at least as far as we are aware,had not taught that the concentration of magnesium acetate was importantfor the RNase III reaction, having stated that it could be used at broadrange of concentrations up to 100 mM. Therefore, our observation thatthere was significant and increasing smearing of the ssRNA bands,particularly at magnesium acetate concentrations of 5 mM and above wassurprising and unexpected. This result showed for the first time that amuch lower divalent magnesium cation concentration than previouslystated was needed in order to maintain the integrity of the ssRNA, andthat the 10 mM concentration which had been used in the art was too highand led to significant degradation of ssRNA. Still further, as shownelsewhere herein, the digestion of dsRNA was incomplete when the RNaseIII treatment was performed using the 10 mM magnesium cationconcentration, which was very surprising because this level of magnesiumcations for RNase III digestion has been taught in the art for about 35years without question or change.

Still further, as shown in EXAMPLE 9, the biological effectiveness ofsingle-stranded modified mRNAs (e.g., pseudouridine-modified mRNAs) forexpression of encoded proteins comprising iPSC reprogramming factors wasgreater if the modified mRNAs encoding the iPSC reprogramming factorswere treated with the RNase III treatment using 2 mM Mg²⁺ rather than 10mM Mg²⁺. Even more surprising, the effectiveness of RNase III-treatedunmodified (GAUC) ssRNAs or mRNAs encoding iPS cell induction factorsthat were treated with the RNase III using 2 mM Mg²⁺ rather than 10 mMMg²⁺ were very different—with no iPS cells being induced using 10 mMMg²⁺, but many iPS cells being induced by the mRNAs that were treatedwith RNase III using 2 mM Mg²⁺ (e.g., EXAMPLE 10).

Example 2: The Effects of Divalent Magnesium Cation Concentration on theCompleteness of RNase III Digestion of dsRNA is Detectable UsingdsRNA-Specific Monoclonal Antibody J2

Different known amounts of a dsRNA substrate were digested with usingthe RNase III treatment in the presence of different concentrations ofdivalent magnesium cations and then the amounts of detectable dsRNAremaining were analyzed by dot blot assays using the dsRNA-specificmonoclonal Antibody J2.

As was previously reported (Leonard et al., 2008), dsRNA stretches of40-bps or more are needed to dimerize TLR3s to elicit an innate immuneresponse. Antibody J2 can recognize dsRNA of 40-bps or more.Accordingly, the J2 monoclonal antibody was chosen because it canrecognize only biologically relevant sizes of dsRNA that will induceinterferon production through activation of TLR3.

The dot blot assay results, as depicted in FIG. 3, show that thedigestion of dsRNA contaminants by RNase III varied with theconcentration of divalent magnesium cations present in the reaction. Inthis case, most of the dsRNA contaminant was digested at a finalconcentration of magnesium acetate less than about 5 mM, and digestionappeared to be complete between about 2 mM and about 4 mM of divalentmagnesium cations.

Example 3: Effect of Mg^(e) Cation Concentration on Completeness ofdsRNA Digestion by RNase III Compositions as Detected UsingdsRNA-Specific Monoclonal Antibody K1

Samples containing different known amounts of dsRNA were treated withRNase III in the presence of varying amounts of divalent magnesiumcations and then analyzed by dot blot assay for the amount of dsRNAremaining using the monoclonal antibody K1 after RNase III treatment.

As discussed in EXAMPLE 2, dsRNA stretches of 40 bps or more are neededto dimerize TLR3s to elicit an innate immune response. Similar to the J2monoclonal antibody, monoclonal antibody can recognize dsRNA of 40-bp ormore. Accordingly, this antibody was chosen because it can recognizeonly biologically relevant dsRNA pieces that will induce interferonproduction through activation of TLR3.

The results, as depicted in FIG. 4, shows that the ability to digestdsRNA contaminants varied based upon the concentration of divalentmagnesium cations used for the RNase III treatment. Using the K1antibody, digestion of the dsRNA contaminant appeared to be almostcomplete at a final concentration of magnesium acetate between about 1mM and 5 mM magnesium acetate, and digestion of the dsRNA appeared to becomplete at between about 2 mM and 4 mM magnesium acetate.

Example 4: Effects of RNase III Treatment on Small (255-Nucleotide or156-Nucleotide) and Large (955-Nucleotide) ssRNA Transcript Integrityand Degree of dsRNA Digestion with Different Concentrations of Mg⁺²

One microgram of the RNA substrate comprising both 1671-basepair dsRNAand 255- and 136-nucleotide ssRNA portions, and a 955-nucleotide ssRNAcontrol transcript were mixed and treated with 20 nanomolar RNase III inreaction buffers containing from 0 to 10 mM magnesium acetate. Ideally,the reaction would digest the 1671-basepair dsRNA portion of the RNAsubstrate and leave the 255-nucleotide and 136-nucleotidesingle-stranded RNA termini of this RNA substrate and the 955-nucleotidessRNA control transcript undigested and intact.

As can be seen from the results in FIG. 5, the ability to digest dsRNAcontaminants while maintaining the integrity of both small and largessRNA varied based upon the concentration of the divalent magnesiumcation present in the reaction. In this case, an optimal dsRNAcontaminant digestion occurred when the final concentration of magnesiumacetate was between about 1 and 4 mM divalent magnesium, and preferably,between about 2 mM and about 3 mM divalent magnesium. At theseconcentrations of divalent magnesium cation, the dsRNA portion of theRNA substrate was approximately completely digested and minimal smearingof the ssRNA bands was observed on the gel, evidence that both ssRNAtranscripts remained preserved and intact.

Example 5: Example of Analyses Performed to Evaluate the Effects of[Mg+2] in the Presence of Different Monovalent Salts, in this Case 200mM Potassium Glutamate, on RNase III Activity on dsRNA and ssRNA,Including Effects on Completeness of dsRNA Digestion and Integrity ofssRNA

One microgram of both dsRNA and ssRNA transcripts was treated with 20nanomolar RNase III in reaction mixture containing 33 mM Tris-acetate,pH 8, 200 mM potassium glutamate (in place of potassium acetate) andvarying concentrations of divalent cation ranging from 0 to 10 mMmagnesium acetate.

As can be seen from the results in FIG. 6, RNase III treatment iscapable of effectively digesting dsRNA contaminants while maintainingthe integrity of the ssRNA using different monovalent salts, in thiscase, potassium glutamate in place of potassium acetate. In the presentEXAMPLE 5, optimal reactions included between about 1 and about 5 mMfinal concentration of magnesium acetate, and more preferably betweenabout 2 and about 4 mM final concentration of magnesium acetate.

Example 6: Effect of RNase III on ssRNA Integrity and Degree of dsRNADigestion Using 1 mM Mg⁺² and Different Concentrations of PotassiumGlutamate

In reactions containing both dsRNA and ssRNA transcripts, theconcentration of potassium glutamate in the reaction was increased from0 to 300 mM final concentration. Each reaction contained 20 nM RNaseIII, 33 mM Tris-acetate, 1 mM magnesium acetate and varying amounts ofpotassium glutamate. As can be seen in FIG. 7, RNase III exhibitssuperior binding patterns and contaminant digestion at specificconcentrations of potassium glutamate salt. At this concentration ofmagnesium acetate, the dsRNA appeared to be approximately completelydigested and the ssRNA was not significantly digested at allconcentrations of potassium glutamate concentrations tested.

Example 7: Effect of the RNase III Treatments of dsRNA or ssRNASubstrates in Separate Reactions Comprising 1 mM Final Concentration ofMg⁺² and Varying Concentrations of Potassium Acetate as the MonovalentSalt

Either dsRNA substrates or a ssRNA substrate were treated in separatereactions with RNase III in reaction mixtures containing 20 nM RNaseIII, 33 mM Tris-acetate, 1 mM magnesium acetate and varying finalconcentrations of potassium acetate between 0 and 300 mM.

As can be seen in FIG. 8, at a final concentration of 1 mM Mg²⁺ cations,RNase III effectively digested the dsRNA substrate, but did not digestthe ssRNA, at all concentration of potassium acetate between 50 and 300mM final concentration. By comparing results such as those shown in thisFIG. 8 and previous FIG. 7, the applicants concluded that a compoundsuch as a monovalent salt is generally needed to maintain ionicstrength, but, provided the final concentration is sufficient (e.g., atleast about 50 mM final concentration), neither the identity nor theconcentration of monovalent salt significantly affects the activity ofRNase III on dsRNA or its specificity for dsRNA. This was surprising andunexpected in view of previous publications in the art which had advisedthat the concentration of monovalent salt was an important variable tooptimize in order to affect the activity and specificity of RNase IIIfor dsRNA. Without being bound by theory, the present applicants believethat the function of the monovalent salt with respect to the RNase IIIdigestion is to maintain sufficient ionic strength to stabilizebasepairing of dsRNA regions in the RNA, so that those dsRNA are notdenatured during the RNase III treatment. As discussed elsewhere herein,contrary to what has previously been taught in the art, the applicantsdiscovered that the final concentration of divalent magnesium cations isvery important for the optimal activity and specificity of RNase III fordsRNA and that the final concentration of magnesium cations for optimalactivity and specificity of RNase III for dsRNA is preferably about 1-4mM, most preferably about 2-3 mM, which is much lower than previouslytaught in the art.

Example 8: Effect of RNase III Treatment on ssRNA Integrity and Degreeof dsRNA Digestion with Increasing Amounts of dsRNA Added to theReaction Mixture

The amount of dsRNA that can be digested in a 10 minute, 37° C.incubation with 20 nM RNase III was sequentially increased from onemicrogram (at a concentration of 20 ng/microliter final) to 20micrograms (400 ng/microliter final). The reaction mixture contained 33mM Tris-acetate, pH 8, 200 mM KOAc and 1 mM magnesium acetate. From theresults in FIG. 9, only 1 microgram to 2 micrograms of dsRNA could bedigested under these reaction conditions. One microgram of ssRNA is usedin the RNase III treatment method described herein in order to assurecomplete digestion of dsRNA avoid any potential for insufficient RNaseIII due to a particular sample containing higher levels of dsRNA.However, those with knowledge will understand that less RNase III can beused and will understand that one could do a similar titration to thatdescribed here in order to determine the amount of RNase III needed forparticular types of samples.

Example 9: Effect of RNase III Treatments in the Presence of DifferentLevels of Divalent Magnesium Cations on Levels of In Vivo Translation ofLuciferase-Encoding mRNA Transfected into BJ Fibroblasts

Firefly luciferase mRNA was treated for 20 minutes with RNase III in areaction mixture containing 33 mM Tris-acetate, pH 8, 200 mM KOAc andeither 2 mM or 10 mM magnesium acetate-based buffer. The RNaseIII-treated mRNA was cleaned up by phenol-chloroform extraction,precipitation using ammonium acetate, and washing with 70% ethanol (asin the RNA Quick Cleanup method described herein) and transfected intohuman BJ fibroblast cells in triplicate wells. Eighteen hourspost-transfection, the cells were lysed and assayed for the amount ofluciferase activity produced. The amount of luciferase activity(measured in relative light units, RLU) was averaged for duplicateassays of the triplicate samples (n=6) and was normalized by the amountof protein in the cell lysate.

As shown in FIG. 10, luciferase mRNA that was treated with RNase IIIusing 2 mM divalent magnesium cations exhibited much higher (˜9 fold)measured luciferase activity compared to luciferase mRNA treated withRNase III using 10 mM divalent magnesium cations. This further showsthat the magnesium concentration used in the art for about 35 years doesnot result in optimal biological activity of RNase III-treated mRNA.Though surprising and unexpected, this result is consistent with ourother findings that use of RNase III to treat ssRNA or mRNA did notdigest dsRNA contaminants as effectively using 10 mM magnesium cations,as taught in the art, as using 1-4 mM magnesium cations. Still further,we found that 1-3 mM, and preferably about 2 mM magnesium cations, ismost effective in digesting dsRNA contaminants, while not significantlydigesting ssRNA. In EXAMPLE 10, we show the critical importance of usingthe discovered low concentrations of magnesium cations for RNase IIItreatments of ssRNA or mRNA that is repeatedly or continuouslyintroduced in human or animal cells in order to induce a biological orbiochemical effect. In EXAMPLE 10, the biological effect isreprogramming of human somatic cells to induced pluripotent stem cells.

Example 10: Effects of RNase III Treatments Using Different Levels ofMg2+ on the Ability of Unmodified Cap1, Poly(A)-Tailed (˜150 Adenosines)mRNAs Encoding iPSC Reprogramming Factors to Reprogram Somatic Cells toInduced Pluripotent Stem Cells (“iPSCs”) in the Absence of an Inhibitorof Innate Immune Response Pathways

In this EXAMPLE 10, we show that mRNAs encoding iPSC reprogrammingfactors, wherein said mRNAs contain only unmodified (GAUC) nucleotidesand do not contain a modified nucleotide that reduces an innate immuneresponse (with the exception of the 5′ terminal cap nucleotidecomprising 7-methylguanine and the 2′-O-methylated 5′ penultimatenucleotide to which the cap nucleotide is joined, both of which togethercomprise the cap1 cap structure) can be used to reprogram mammaliansomatic cells to iPSCs without use of an innate immune responseinhibitor such as B18R protein provided that the mRNAs are treated usingthe RNase III treatment methods described herein. Thus, in thisexperiment, we used an mRNA reprogramming factor mix comprisingunmodified mRNAs encoding OCT4, SOX2, KLF4, LIN28, NANOG and cMYC(T58A)in a 3:1:1:1:1:1 molar ratio, wherein the mRNAs were treated with RNaseIII in the presence of 200 mM potassium acetate as a monovalent salt,and either 1, 2, 3, 4, 5 or 10 mM magnesium acetate in order to evaluatethe importance of the divalent magnesium cation concentration in theRNase III treatment step for induction of iPSCs. In another aspect ofthis experiment, the mRNAs were treated with RNase III in the presenceof 200 mM potassium glutamate to evaluate the effects of this monovalentsalt in place of potassium acetate, and either 2 or 10 mM magnesiumacetate in order to evaluate the importance of the divalent magnesiumcation concentration in the RNase III treatment step for induction ofiPSCs.

Methods for using feeder cells and plating BJ fibroblasts forreprogramming and for using TransIT™ (Mirus Bio) transfection reagentfor reprogramming were as described above. Briefly, 1.25×10⁵ BJfibroblast cells were plated on 5×10⁵ NuFF feeder cells. The cells weretransfected daily for 13 days with 1.2 micrograms of a 100 ng permicroliter mRNA reprogramming mix comprising a 3:1:1:1:1:1 molar ratiocontaining OCT4, SOX2, KLF4, LIN28, NANOG and cMYC(T58A). Thetransfection was performed with 2.4 microliters of each Mirus BioTransIT™ Boost and TransIT™ Transfection Reagent as previouslydescribed. The PLURITON™ medium, with 1× penicillin/streptomycin, 1×PLURITON™ supplement and 0.5 U/ml SCRIPTGUARD™ RNase Inhibitor waschanged daily before the cells were transfected. On day 13, the cellswere fixed, immunostained for the alkaline phosphatase iPSC marker, andthe number of alkaline phosphatase-stained iPSC colonies were countedfor each treatment.

As shown in the Table 1 below, the numbers of alkalinephosphatase-positive iPS cells induced in the cells transfected oncedaily with mRNAs that were treated with RNase III in the presence of 1or 2 mM magnesium acetate were much higher than in cells transfectedonce daily with mRNAs treated with higher concentrations of magnesiumacetate. In particular, no alkaline phosphatase-positive cells wereinduced in BJ fibroblast cells that were transfected once daily with themRNA reprogramming mix comprising mRNAs that were treated with RNase IIIin the presence of 10 mM magnesium acetate in the presence of eitherpotassium acetate or potassium glutamate as the monovalent salt.

TABLE 1 Ability of mRNAs treated with RNase III in the presence ofdifferent Mg²⁺ concentrations to generate alkaline phosphatase-positiveiPSCs following repeated transfections of BJ fibroblasts. MonovalentMg(OAc)₂ Number of Alkaline Potassium Salt ConcentratonPhosphatase-positive Used for RNase III Used for RNase III iPSC ColoniesTreatment Treatment on Day 13 Acetate 1 mM 110 Acetate 2 mM 70 Acetate 3mM 3 Acetate 4 mM 3 Acetate 5 mM 2 Acetate 10 mM  0 Glutamate 2 mM 27Glutamate 10 mM  0

These results demonstrate that the RNase III treatment methods describedherein, comprising treating in vitro-transcribed RNA with RNase III inthe presence of about 1-4 mM Mg²⁺, removed dsRNA to a sufficient extentto enable reprogramming of fibroblasts to iPSCs following repeatedtransfections of human BJ fibroblast somatic cells with this mRNAreprogramming mix comprising 6 different mRNAs encoding differentprotein reprogramming factors. In the presence of 1 or 2 mM Mg²⁺, theRNase III treatment very effectively removed dsRNA from an mRNAreprogramming mix so that reprogramming of the BJ fibroblast somaticcells were efficiently reprogrammed to alkaline phosphatase-positivededifferentiated cells or induced pluripotent stem cells. In contrast,an mRNA reprogramming mix comprising the same mRNAs treated using 10 mMMg²⁺, the concentration first recommended by Robertson et al. (RobertsonH D et al., 1968) and believed to be subsequently used as the standardconditions by other researchers since that time, did not result inreprogramming of the BJ fibroblast somatic cells to alkalinephosphatase-positive dedifferentiated cells or induced pluripotent stemcells under otherwise the same conditions. The immunostainingdifferences were also supported by morphological differences observedbetween the cells treated with 1-2 mM compared to 10 mM Mg²⁺. Forexample BJ fibroblasts transfected daily with mRNAs treated with RNaseIII in 2 mM Mg²⁺ exhibited iPSC colonies, whereas BJ fibroblaststransfected daily with mRNAs treated with RNase III in 10 mM Mg²⁺ didnot exhibit a new morphology.

The present researchers believe successful reprogramming of human oranimal somatic cells to iPSC cells using only unmodified ssRNA has notpreviously been reported or demonstrated. Without being bound by theory,we believe that others have not been successful in reprogramming humanor animal cells with unmodified ssRNAs because they have not recognizedthe importance of purifying or treating in vitro-synthesized ssRNA inorder to make ssRNAs that are at least practically free of dsRNA, and,even if they had recognized the importance and benefits of making ssRNAsthat are at least practically free of dsRNA, they have not understood ordeveloped a method for sufficiently purifying or treating said ssRNAs inorder to make them at least practically free of dsRNA, and morepreferably, extremely free or even absolutely free of dsRNA. Forexample, the present researchers have discovered simple, rapid andefficient methods for treating ssRNAs with a double-strand-specificRNase that results in ssRNAs that are at least practically free ofdsRNA. One example of such a double-strand-specific RNase that can beused for this purpose is the endoribonuclease, RNase III. The presentresearchers also discovered, surprisingly and unexpectedly, that amethod for using RNase III that was reported in the literature to removedsRNA from ssRNA to remove the inhibitory activity of dsRNA on in vitrotranslation did not sufficiently remove dsRNA from ssRNAs so that thessRNAs treated using that method could be used for translation in livingcells or for reprogramming living human or animal cells from one stateof differentiation to another state of differentiation (e.g., forreprogramming human or animal somatic cells to iPS cells). In fact,attempts by the present researchers to use ssRNAs that had been treatedwith RNase using the method in the literature for repeated transfectionsto generate iPSCs ultimately resulted in the death of those cells. Stillfurther, not only did the method for using RNase III to remove dsRNA forin vitro applications not work for in vivo applications (and resulted inapoptosis of the cells transfected with ssRNAs so treated), but themethod also degraded the ssRNAs that the present researchers desired tobe translated in the living cells. In other words, not only did theRNase III method in the literature fail to sufficiently remove theundesired dsRNA, it also destroyed a portion of the desired ssRNAs thatencode the proteins of interest. Next, the present researchers tried tomodify all of the conditions that the authors of the RNase III methodfor making ssRNA for in vitro applications, unfortunately to no avail.Thus, although the authors of the existing method suggested thatincreasing the concentration of the monovalent salt in the RNase IIIreaction to a concentration that was higher or lower than what theysuggested might be beneficial, the present inventors tried this withoutsuccess. They also tried multiple different monovalent salts and variedtheir concentrations, but this also did not result in sufficient removalof the dsRNA for the ssRNAs to be used for reprogramming living cells,did not sufficiently reduce the toxicity of the ssRNAs, and stilldamaged or destroyed at least a portion of the desired ssRNAs. Thechange of other variables suggested by the authors of the publishedmethod also did not accomplish the intended goal. Without being bound bytheory, the present researchers believe that the difficulty was due tothe extremely low levels of dsRNA that can be detected by the innateimmune response and other RNA sensors that are present in human andanimal cells to protect those cells from infection by dsRNA viruses andother pathogens. Thus, due to the extreme sensitivity of human or animalcells to dsRNA that is introduced into those cells, a method that issuitable for reducing dsRNA from ssRNAs for use of the ssRNA for invitro applications is not sufficient for making ssRNAs for introducinginto living human or animal cells. Still further, those innate immuneresponse and other RNA sensors (e.g., toll like receptors, e.g., TLR3,interferons, and other such sensors) are induced to higher levels ifdsRNAs are introduced into said cells. In other words, if ssRNAs thatare introduced into living human or animal cells contain even a minutequantity of contaminating dsRNA, that dsRNA induces innate immuneresponse and other RNA sensors to respond, which can cause toxicity andinhibition of protein synthesis in said cells. The initial response maysensitize the cells to be even more responsive to subsequent repeatedintroductions of the ssRNAs into the cells, causing further toxicity andinhibition of protein synthesis (e.g., Kalal M et al. 2002; Stewart II,W E et al. 1972). If prolonged, these effects lead to increasingtoxicity, and cell death. Thus, with respect to certain prior artmethods for reprogramming human somatic cells to iPS cells, the innateimmune response and other RNA sensor responses are induced each time thessRNAs encoding reprogramming factors are introduced into the cells. Forexample, some of the molecules that are induced and activated by dsRNAare interferons, which can inhibit protein synthesis, inducecytotoxicity, and if prolonged, result in cell death.

Example 11. Feeder-Free Reprogramming of Human Somatic Cells to iPSCells on MATRIGEL™ GFR Matrix Using Single-StrandedPseudouridine-Containing mRNAs Encoding iPSC Induction Factors in theAbsence of an Inhibitor or Agent that Reduces the Expression of anInnate Immune Response Pathway Materials and Methods for Example 11.

In EXAMPLE 11 embodiments, each in vitro-synthesizedpseudouridine-containing ssRNA (i.e., synthesized using ψTP in place ofUTP in the IVT reaction) that encoded an iPSC induction factor [e.g.,OCT4, SOX2, KLF4, LIN28, and either cMYC or cMYC(T58A)] or otherpseudo-uridine-containing ssRNAs transfected together with the iPSCinduction factors was treated with RNase III with 1 mM Mg(OAc)₂ prior tocapping and tailing of the ssRNA. In this EXAMPLE 11, the RNAse IIItreatment reactions also contained 0.8 U/microliter SCRIPTGUARD™ RNaseinhibitor (CELLSCRIPT, INC.).

Feeder-Free Reprogramming of Human Fibroblast Cells to iPSC Cells UsingSingle-Stranded mRNA iPSC Induction Factors

Prior to use for reprogramming, BJ fibroblasts (ATCC) were plated 5×10⁴cells per well on 6-well tissue culture plates coated with 83 ng perwell of MATRIGEL™ GFR matrix (BD Biosciences, San Jose, Calif.) inAdvanced MEM (Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS(Fisher) and 2 mM GLUTAMAX™-I (Invitrogen, Carlsbad, Calif.), a minimumessential medium (MEM) useful for growth of fibroblast cells.

On the following day, the medium was changed to a “Feeder-freeReprogramming Medium” developed by the present applicants. ThisFeeder-free Reprogramming Medium was composed of Dulbecco's modifiedEagle medium with nutrient mixture F-12 (DMEM/F12; (DMEM/F12;Invitrogen, Carlsbad, Calif.) supplemented with 20% KNOCKOUT™ serumreplacement (Invitrogen), 2 mM GLUTAMAX™-I (Invitrogen), 0.1 mMnon-essential amino acids solution (Invitrogen), 2 micromolar of thetransforming growth factor β (TGFβ) inhibitor STEMOLECULE™ SB431542(Stemgent®, Cambridge, Mass., USA), 0.5 micromolar of the MEK signalingpathway inhibitor STEMOLECULE™ PD0325901 (Stemgent), and/or 10 ng/ml therecombinant mouse cytokine, leukemia inhibitory factor (LIF or mLIF;Invitrogen, Carlsbad, Calif.), and 100 ng/ml basic human recombinantfibroblast growth factor (FGF; Invitrogen) with penicillin-streptomycinantibiotics. In some experiments, a lower or higher concentration of oneor more of these inhibitors is used (e.g., 1-20 micromolar of the TGFβinhibitor, 0.5-10 micromolar of the MEK signaling inhibitor, and/or 5-50ng/ml the recombinant mouse cytokine, leukemia inhibitory factor). Sincesome of the molecules being inhibited may be introduced by the reagentsor media used (e.g., TGFβ in the MATRIGEL™ or other extracellularmatrix, the concentrations of the inhibitors used may vary based on thereagents and media used. In some experiments, the TGFβ inhibitor, MEKsignaling inhibitor, and/or LIF was omitted from the feeder-freereprogramming medium. The Feeder-free Reprogramming Medium was changeddaily one hour prior to each transfection with mRNA reprogrammingfactors. Cells were transfected daily for 18 consecutive days using theTRANSIT™ mRNA transfection kit (Mirus Bio LLC, Madison, Wis., USA) asdescribed in the product literature: Briefly, a solution comprising amixture of all of the mRNA reprogramming factors was diluted with 250microliters of OPTI-MEM® I reduced serum medium (Invitrogen, Carlsbad,Calif.), and then 2.4 microliters of TRANSIT™ BOOST was added and mixed,followed by 2.4 microliters of the TRANSIT™ transfection reagent. Insome embodiments, no inhibitor of expression of an innate immuneresponse pathway was used. In some other embodiments, 132 ng ofpseudouridine-containing mRNA encoding the B18R protein was added to thereprogramming factors comprising mRNA encoding OCT4, SOX2, KLF4, LIN28,and cMYC for reprogramming BJ fibroblasts. This transfection mix wasapplied dropwise to cells. Cells were then incubated at 37° C. in 5% CO₂until the next day's transfection. After 18 transfections, the mediumwas changed to a different “iPSC Maintenance Medium” that that wascomposed of DMEM/F12 supplemented with 20% KNOCKOUT™ serum replacement,1 mM L-glutamine, 0.1 mM non-essential amino acids solution, and 100ng/ml basic human recombinant fibroblast growth factor (FGF) (all fromInvitrogen, Carlsbad, Calif.) with penicillin-streptomycin antibioticsfor a few more days until iPSC colonies were big enough to pickmanually.

In another experiment to evaluate the effect of using differentconcentrations of the TGFβ inhibitor, STEMOLECULE™ SB431542,reprogramming of BJ fibroblasts was performed as described above exceptthat the concentration of the TGFβ inhibitor STEMOLECULE™ SB431542 wasused in the reprogramming medium at a concentration of either 0, 1, 2,or 4 micromolar, and, in this experiment, the BJ fibroblast cells weretransfected for only 17 consecutive days rather than for 18 days.

In another experiment to evaluate the effect of using differentconcentrations of the MEK inhibitor STEMOLECULE™ PD0325901,reprogramming of BJ fibroblasts was performed as described above with a2 micromolar concentration of the TGFβ inhibitor, and the MEK inhibitorSTEMOLECULE™ PD0325901 was used in the reprogramming medium at aconcentration of 0, 0.5, 1, 2, 10, or 15 micromolar, and, in thisexperiment, the BJ fibroblast cells were transfected for only 17consecutive days rather than for 18 days.

Maintenance of iPSC Colonies Generated from Feeder-Free Reprogramming ofHuman Somatic Cells Using Single-Stranded mRNA iPSC Induction Factors

The iPSC colonies resulting from feeder-free reprogramming were manuallypicked and transferred into 12-well plates coated with 42 ng per well ofMATRIGEL™ GFR matrix (BD Biosciences) containing a medium composed ofone-half mTESR®-1 medium (StemCell Technologies, Vancouver, BC, Canada)and one-half of the above-described iPSC Maintenance Medium with 10micromolar Y27632 STEMOLECULE™ ROCK inhibitor (Stemgent), acell-permeable small molecule inhibitor of Rho-associated kinases.Plates were incubated at 37° C. in 5% CO₂ overnight, after which iPSCcolonies were again manually picked and maintained in mTESR medium(StemCell Technologies). In order to expand the cultures, the iPSCcolonies were passaged in dispase solution (1 mg/ml) in DMEM/F12 medium(StemCell Technologies, Vancouver, BC, Canada) in 6-well MATRIGEL™ GFRmatrix-coated plates (BD Biosciences); the iPSCs were incubated in thedispase solution for 7 minutes at 37° C. and 5% CO₂, washed three timeswith 3 mls of DMEM/F12 medium, removed in mTESR®-1 medium (StemCellTechnologies), and plated into wells of a fresh MATRIGEL™ GFRmatrix-coated plate (BD Biosciences) at appropriate split ratios.

Immunocytochemistry of iPSC Colonies

Cells were washed twice in 1× phosphate-buffered solution (PBS) andfixed in 4% paraformaldehyde in PBS at room temperature for half anhour. After 3 washes in 1×PBS, cells were washed 3 times in wash buffer(0.1% Triton-X100 in PBS), and blocked for one hour at room temperaturein blocking solution (0.1% Triton-X100, 1% BSA, 2% FBS in PBS). Primaryantibodies were diluted 1 to 1,000 in blocking solution and applied tocells overnight at 4° C. Cells were washed 6 times in wash buffer andsecondary antibodies, diluted 1 to 1,000 in blocking buffer, wereapplied for 2 hours at room temperature in the dark. After 6 washes withwash buffer, cells were washed twice in 1×PBS before imaging.

Protocol for Differentiation of Feeder-Free Reprogrammed iPSCs toCardiomyocytes

Induced pluripotent stem cell colonies were dissociated with TrypLESelect (Invitrogen, Carlsbad, Calif.) for 5 minutes at 37° C. in 5% CO₂.TrypLE was neutralized in 1:1 ratio with mTESR supplemented with 10micromolar Y27632 ROCK inhibitor (Stemgent) and 25 micrograms/mlgentamicin (Invitrogen, Carlsbad, Calif.), spun down, and resuspended inthe same medium. Dissociated iPSCs were seeded 5×10⁶ cells in ultra lowattachment T25 flasks (Corning Life Sciences, Lowell, Mass.) andincubated overnight at 37° C. in 5% CO₂. The next day, media wasexchanged to 50% mTESR and 50% aggregate transition medium, DMEMGLUTAMAX™ (Invitrogen, Carlsbad, Calif.), 10% FBS (Fisher), 50 ng/mlFGFb (Invitrogen, Carlsbad, Calif.), and 25 micrograms/ml gentamicin(Invitrogen, Carlsbad, Calif.), and the aggregates were split into 2ultra low attachment T25 flasks. For the following 12 days aggregateswere fed cardiac induction medium, DMEM GLUTAMAX™ (Invitrogen, Carlsbad,Calif.), 10% FBS (Fisher), 50 ng/ml FGFb (Invitrogen, Carlsbad, Calif.).After aggregates began to beat, media was changed to cardiac maintenancemedia, DMEM low glucose (Invitrogen, Carlsbad, Calif.), 10% FBS, 25micrograms/ml gentamicin.

Results for Example 11.

BJ fibroblasts plated on MATRIGEL™ GFR matrix were transfected daily for18 consecutive days with pseudouridine-containing mRNA reprogrammingfactors encoding OCT4, SOX2, KLF4, LIN28, and cMYC or cMYC(T58A) inFeeder-free Reprogramming Medium. The ssRNA reprogramming factors wascomposed of pseudouridine-containing mRNAs prepared as described aboveand in the literature provided with the T7 mSCRIPT™ standard mRNAproduction system (CELLSCRIPT, INC., Madison, Wis., USA), except thatpseudouridine 5′ triphosphate (ΨTP) was substituted for uridine 5′triphosphate (UTP), and, prior to capping or polyadenylation, the invitro-transcribed RNAs were treated using the RNase III treatment asdescribed herein with a concentration of 1 mM Mg acetate. No feedercells were used. Unless otherwise specifically stated, no B18R proteinor other inhibitor or agent that reduces the expression of an innateimmune response pathway was used. The cells survived and grew toconfluence, and by the end of the transfection regimen, were actuallyover confluent. In experiments using pseudouridine-containing mRNA thatencoded the cMYC(T58A) mutant of the cMYC protein, iPSC colonies beganto appear around day 14, after 15 transfections (FIG. 11), based on thefirst day of transfection being counted as day 0. In experiments, usingmRNA that encoded the wild-type long version of the cMYC protein, iPSCcolonies began to appear around day 16. In this experiment, iPSCcolonies were obtained only when LIF protein or a TGFβ or MEK smallmolecule inhibitor (e.g., SB431542 TGFβ inhibitor or PD0325901 MEKinhibitor) was present in the medium (FIG. 12). No iPSC colonies formedin the absence of these inhibitors.

In other mRNA reprogramming experiments using PLURITON™ mRNAreprogramming medium (Stemgent) on MATRIGEL™ GFR matrix without feedercells, massive cell death was observed in the first week oftransfections with the same pseudouridine-containing mRNA reprogrammingfactors encoding OCT4, SOX2, KLF4, LIN28, and the wild-type long versionof cMYC; all of the BJ fibroblast cells died in PLURITON™ medium and noiPSC colonies were observed. Using the pseudouridine-containing mRNAencoding OCT4, SOX2, KLF4, LIN28, and the cMYC(T58A) mutant in theabsence of the small molecules, SB431542(TGFβ inhibitor), PD0325901 (MEKInhibitor), and LIF (leukemia inhibitor factor), a majority of the cellsdied in PLURITON™ medium, but a small number of surviving cells wereable to form iPSC colonies after 18 transfections. However, many feweriPSC colonies were generated from feeder-free BJ fibroblasts inPLURITON™ medium than were generated in feeder-free BJ fibroblaststransfected with the same pseudouridine-containing mRNA reprogrammingfactors encoding OCT4, SOX2, KLF4, LIN28, and the cMYC(T58A) in theFeeder-free Reprogramming Medium supplemented with SB431542 TGFβinhibitor, PD0325901 MEK inhibitor, and LIF, as described in the presentExample (e.g., see FIG. 12). iPSC colonies that formed on MATRIGEL™ GFRmatrix in the Feeder-free Reprogramming Medium and developed asdescribed in the present Example stained positive for an alkalinephosphatase, characteristic of iPSC colonies (FIG. 12). After a coupleof days in iPSC maintenance medium, iPSC colonies were manually picked,plated into fresh MATRIGEL™ GFR matrix-coated plates, and expanded.Cultures of each colony grew and could be expanded as expected foriPSCs, needing to be passaged every 3 to 4 days via splitting in dispasesolution, and having been kept in culture for at least 10 passages todate. Cells from the colonies also stained positive for iPSCpluripotency markers: NANOG, TRA-1-60, SSEA4, OCT4, and SOX2 (e.g., FIG.13). The immunostaining results shown in FIG. 13 are from an iPSC cellline established from an iPSC colony picked from a reprogrammingexperiment for reprogramming BJ fibroblasts to iPS cells, wherein 132 ngof pseudouridine-containing mRNA encoding the B18R protein was added tothe reprogramming factors comprising pseudouridine-containing mRNAsencoding OCT4, SOX2, KLF4, LIN28, and cMYC. Other iPSC cell linesinduced in the absence of mRNA encoding B18R protein or using otherconditions described in this Example also stain positively for iPSCpluripotency markers.

It was previously determined that 1×10⁴ human BJ fibroblast cells was anoptimal cell density per well in a 6-well dish for successful iPSCinduction on feeder cells. Initial reprogramming experiments indicatedthat a cell density of 1×10⁴ BJ fibroblast cells per well was notsufficient for generating as many iPSC colonies from feeder cell-freereprogramming of the BJ fibroblasts on MATRIGEL™ GFR matrix as weregenerated from reprogramming using feeder cells. However, feedercell-free reprogramming of higher numbers of BJ fibroblasts to iPSCcolonies was achieved when the cell density per well of the BJfibroblasts was increased to 5×10⁴ cells per well on MATRIGEL™ GFRmatrix.

Table 2 below shows the number of iPSC colonies counted on Day 18 whenBJ fibroblasts were transfected daily for 18 days with the mixture ofpseudouridine-containing mRNAs encoding the five iPSC induction factors:OCT4, SOX2, KLF4, LIN28, and cMYC(T58A), as described above, and,additionally, with or without pseudouridine-containing mRNA encodingB18R protein, and plated at a cell density of 5×10⁴ cells per well onMATRIGEL™ GFR matrix in the iPSC Feeder-free Reprogramming Medium thatwe developed as described above, or at a cell density of 1×10⁴ cells onhuman neonatal fibroblast feeder cells in the Feeder-free ReprogrammingMedium. The new iPS Feeder-free Reprogramming Medium used forreprogramming of the feeder-free cells on the MATRIGEL™ GFR matrix alsocontained the TGFβ inhibitor STEMOLECULE™ SB431542, the MEK inhibitorSTEMOLECULE™ PD0325901, and the LIF protein as described above.

TABLE 2 Feeder-free Reprogramming of BJ Fibroblasts to iPSCs..Pseudouridine- containing mRNA Number of Encoding B18R iPSC ColoniesSubstrate Protein Used Observed Feeder-free MATRIGEL matrix YES 72Feeder-free MATRIGEL matrix NO 102 Human neonatal fibroblast feeders YES81 Human neonatal fibroblast feeders NO 76

Table 3 below shows the number of iPSC colonies counted on Day 17 whenBJ fibroblasts were transfected daily for 17 days with the mixture ofpseudouridine-containing mRNAs encoding the five iPSC induction factors:OCT4, SOX2, KLF4, LIN28, and cMYC(T58A) in the presence of the indicatedconcentrations of the TGFβ inhibitor STEMOLECULE™ SB431542 (Stemgent).

TABLE 3 Feeder-free Reprogramming of BJ Fibroblasts in the Presence ofDifferent Concentrations of the TGFβ inhibitor STEMOLECULE ™ SB431542(Stemgent). TGFβ inhibitor SB431542 iPSC Colonies Concentration (μM)Observed 0 0 1 9 2 71 4 160

Table 4 below shows the number of iPSC colonies counted on Day 17 whenBJ fibroblasts were transfected daily for 17 days with the mixture ofpseudouridine-containing mRNAs encoding the five iPSC induction factors:OCT4, SOX2, KLF4, LIN28, and cMYC(T58A) in the presence of 2 micromolarof the TGFβ inhibitor STEMOLECULE™ SB431542 (Stemgent) and the indicatedconcentrations of the MEK inhibitor STEMOLECULE™ PD0325901 (Stemgent).

TABLE 4 Feeder-free Reprogramming of BJ Fibroblasts in the Presence ofDifferent Concentrations of the MEK inhibitor STEMOLECULE ™ PD0325901(Stemgent). PD0325901 iPSC Colonies Concentration (μM) Observed 0 1 0.577 1 27 2 94 10 11 15 0

Differentiation of Feeder-Free Reprogrammed iPSCs to Cardiomyocytes

Induced pluripotent stem cells that were generated from feeder-freereprogramming of BJ fibroblasts using OCT4, SOX2, KLF4, LIN28, and cMYCpseudouridine-containing mRNA reprogramming factors andpseudouridine-containing mRNA encoding B18R differentiated into beatingaggregates of cardiomyocyte cells using the protocol for cardiomyocytedifferentiation as described in the Materials and Methods for EXAMPLE11. Beating cardiomyocyte aggregates were first observed after 13 days.Videos of the beating cardiomyocyte aggregates were recorded.

Example 12. Studies on Variables Affecting Efficiency of mRNAs EncodingiPSC Induction Factors to Reprogram Human BJ Fibroblasts to iPS CellsUsing Feeder Layers Materials and Methods for EXAMPLE 12.

Methods for Using Feeder Cells and Plating BJ Fibroblasts forReprogramming to iPSCs with mRNA iPSC Induction Factor.

Nuffs feeder cells and plating of BJ fibroblasts were done as describedin the General Materials and Methods section. The mRNA reprogrammingfactors encoding OCT4, SOX2, KLF4, LIN28 and MYC (e.g., either c-MYC,c-MYC(T58A), or L-MYC) were prepared in a 3:1:1:1:1 molar ratio asdescribed previously. Unless otherwise stated the ssRNAs were treatedwith RNase III using RNase III treatment as described herein with 1 mMor 2 mM magnesium acetate described herein.

RNAiMAX™ mRNA Transfection Protocol

BJ fibroblast media from BJ fibroblasts plated on Nuff feeder cells wasremoved and added to PLURITON™ mRNA reprogramming media (Stemgent,Cambridge, Mass.) (base media with supplement andpenicillin/streptomycin) (2 mls) and 4 microliters of B18R recombinantprotein (EBiosciences, San Diego, Calif.) was added to a finalconcentration of 200 ng/ml. The cells were incubated at 37° C. under 5%CO₂ for 4 hours before transfecting the mRNA. To transfect the BJfibroblasts with 3:1:1:1:1 mRNA mix (OCT4, SOX2, KLF4, LIN28) andc-MYC(T58A) or cMYC, 12 microliters of the 100 ng/μl RNA mixture (1.2micrograms total) was added to 48 microliters of OptiMEM™ medium(Invitrogen, Carlsbad, Calif.) in tube A. In some experiments, mRNAencoding EGFP protein was also added to make a 3:1:1:1:1:1 mRNA mix ofOCT4, SOX2, KLF4, LIN28, c-MYC(T58A) and EGFP, and in some experiments,the total microgram amount of the mRNA mixture used for transfection wasvaried, as indicated for that experiment. In tube B, 54 microliters ofOptiMEM medium was mixed with 6 microliters of RNAiMAX™ (Invitrogen,Carlsbad, Calif.). Five microliters of RNAiMAX™ was used for each 1microgram of total RNA used for a transfection. Tube A was mixed withtube B for 15 minutes at room temperature and then the mix was added tothe 2 mls of PLURITON medium already on the BJ fibroblasts plated onNuffs. Unless otherwise indicated, the medium was changed 4 hours aftereach RNAiMAX™ transfection with new PLURITON medium with or without B18Rprotein at 200 ng/ml and incubated overnight at 37° C. in 5% CO₂. On thefollowing day, the transfection mix was made in the same way asdescribed above and the mRNA/RNAiMAX™ complexes were added to the mediumalready in each well without changing the medium prior to adding themRNA/RNAiMAX™ complexes. The media were again changed 4 hours after thetransfections and B18R protein was added at 200 ng/ml and the cells wereincubated overnight at 37° C. in 5% CO₂. Nuff-conditioned PLURITONmedium was used to replace PLURITON media on the sixth day oftransfections. These transfections were repeated every day at the sametime for 16 additional mRNA transfections for a total of 18 mRNAtransfections.

However, in other experiments, as indicated in the RESULTS section, themedium was changed before every transfection with the mRNA mixturesencoding the iPSC induction factors and, in some cases, with or withoutadditional mRNAs encoding other proteins, or, in some experiments, themedium was not changed 4 hours after the RNAiMAX™ transfections.

TransIT™ mRNA Transfection Protocol

BJ fibroblast media from BJ fibroblasts plated on Nuff feeder cells wasremoved and added to PLURITON™ mRNA reprogramming media (Stemgent,Cambridge, Mass.) (base media with supplement andpenicillin/streptomycin) (2 mls) and 4 microliters of B18R recombinantprotein (EBiosciences, San Diego, Calif.) was added to a finalconcentration of 200 ng/ml. The media can be changed immediately beforeeach transfection with Mirus mRNA Transfection Reagent (Mirus Bio,Madison, Wis.). To transfect the BJ fibroblasts with 3:1:1:1:1 mRNA mix(OCT4, SOX2, KLF4, LIN28) and c-MYC(T58A) or cMYC, the mRNA mix (0.6 to1.4 micrograms of total mRNA) was added to 120 microliters of OptiMEM[without TransIT Boost™ (Mirus Bio), TransIT and the mRNA mix volume]and then TransIT Boost (2 microliters per microgram of mRNA) and TransITmRNA transfection reagent (2 microliters per microgram of mRNA) weremixed with the mRNA. The mRNA-TransIT mix was incubated for 2 minutesand then added to each well of BJ fibroblasts on Nuff feeders inPLURITON medium. The following day, the PLURITON media were changedbefore transfecting the same dose of mRNA using TransIT Boost andTransIT mRNA transfection reagent. Nuff-conditioned PLURITON medium wasreplaced by PLURITON medium on the sixth day of transfections. A totalof 18 transfections were performed.

Embryoid Body Spontaneous Differentiation Protocol

The embryoid body spontaneous differentiation protocol was performed aspreviously described (Huangfu et al., 2008), and as summarized in theGeneral Materials and Methods.

Live Cell Staining of iPSC Colonies with Tra-1-60

TRA-1-60 is considered to be one relatively stringent marker of fullyreprogrammed iPS cells (Chan et al. 2009). The Tra-1-60 live cellimaging was done with the StainAlive Dylight 488 Mouse anti-HumanTra-1-60 antibody (Stemgent) according to the manufacturer'sspecifications.

Results for Example 12.

RNase III Treatment Reduced the Level of dsRNA.

The in vitro-transcribed RNAs used in these experiments were all treatedwith RNase III to remove dsRNA prior to capping and tailing reactions(FIG. 14). RNase III treating of the Ψ-mRNAs (or Ψ- and m5C-mRNAs, datanot shown)resulted in undetectable levels of dsRNA that was recognizedby the monoclonal dsRNA antibody J2 in dsRNA dot blot experiments whenless than or equal to 1 microgram of RNA was spotted on the membrane(e.g., FIG. 14). We believed that removing dsRNA contaminants from ourmRNAs would greatly reduce overall toxicity and therefore enhancecellular reprogramming when mRNAs were transfected for up to 18 straightdays. To further reduce any potential innate immune reactivity to ourmRNAs, we also incorporated pseudouridine (Ψ) in place of conventionaluridine (and in some of our mRNAs, also 5-methylcytidine (m⁵C) in placeof cytidine); Drs. Kariko and Weissman and their co-workers (Kariko etal., 2005; Kariko et al., 2008; Kariko and Weissman, 2007) have shownthat mRNAs containing these non-canonical nucleosides exhibitsignificantly reduced cellular immune responses.

iPSC Induction from BJ Fibroblasts Using RNAiMAX™ Protocols

When a 3:1:1:1:1 molar ratio of RNase III-treated Ψ- and m5C-mRNAsencoding OCT4, SOX2, KLF4, LIN28 and c-MYC or c-MYC(T58A) wereintroduced into BJ fibroblasts (grown on irradiated human Nuff feedercells) using the RNAiMAX™ transfection reagent each day for 18 days,mesenchymal-to-epithelial transformation became obvious by day 12 oftransfections, and tightly packed epithelial colonies with highnuclear-to-cytoplasmic ratios were generated by day 16 of transfections.BJ fibroblasts transfected with total daily doses of 1.2 micrograms mRNAshowed Tra-1-60 positive colonies on day 18, both for the cellstransfected with mRNA encoding c-MYC and for the cells transfected withmRNA encoding the c-MYC(T58A) mutant protein (FIG. 15).

Table 5 shows the relative numbers of iPSC colonies observed that wereTra-1-60-positive on day 18 of transfection when BJ fibroblasts weretransfected using RNAiMAX™ with different doses of the RNase III-treatedmRNAs encoding OCT4, SOX2, KLF4, LIN28 and c-MYC or c-MYC(T58A) thatcontained both Ψ and m⁵C modified nucleosides.

TABLE 5 iPSC induction from BJ fibroblasts using RNase III-treated Ψ-and m⁵C-modified mRNAs encoding OCT4, SOX2, KLF4, LIN28 and c-MYC orc-MYC(T58A) complexed with RNAiMax ™. No. of iPSC Colonies OnReprogramming Day 18 Treatment (Tra-1-60 positive) Untreated 0 MockTransfected 0 1.2 μg 5 Factors (cMYC) 6 1.2 μg 5 Factors (cMYC) 3 (+B18R200 ng/ml) 0.6 μg 5 Factors (cMYC) 0 0.3 μg 5 Factors (cMYC) 0 1.2 μg 5Factors (cMYC T58A) 38 1.2 μg 5 Factors (cMYC T58A) 20 (+B18R 200 ng/ml)0.6 μg 5 Factors (cMYC T58A) 3 0.3 μg 5 Factors (cMYC T58A) 0

mRNA mixes with c-MYC (T58A) in place of wild type c-MYC showed ˜6 foldmore colonies at the 1.2 micrograms mRNA dose and even resulted inTra-1-60-positive colonies at the 0.6-microgram dose. Transfection mixescontaining wild-type c-MYC did not result in any WPS colonies at the0.6-microgram dose (Table 5). Addition of the B18R recombinant proteindid not aid in reprogramming efficiency in this experiment and it evenappeared to be detrimental, since it resulted in about half theTra-1-60-positive iPSC colonies compared to wells without B18R protein(Table 5).

When the RNAiMAX™ transfection protocol was repeated with only the3:1:1:1:1 molar ratio of RNase III-treated Ψ- and m⁵C-modified mRNAsencoding OCT4, SOX2, KLF4, LIN28 and c-MYC, we again saw a reduction inthe number of colonies morphologically resembling iPS colonies when B18Rprotein was used (Table 6). Without being bound by theory, it ispossible that B18R was not beneficial in this experiment because theRNase III-treated Ψ- and m⁵C-modified mRNAs did not elicit a substantialinnate immune response.

TABLE 6 iPSC induction from BJ fibroblasts ± B18R Protein using RNaseIII-treated Ψ- and m⁵C-modified mRNAs encoding OCT4, SOX2, KLF4, LIN28and c-MYC complexed with RNAiMax ™. Amount of mRNA Mix Used No. of iPSCfor Transfection Using Additional Colonies (based on RNAiMAX ™ Treatmentcell morphology) None - Mock Transfection None 0 1.2 μg mRNA mix None 201.2 μg mRNA mix +B18R Protein 8 0.8 μg mRNA mix None 5 0.8 μg mRNA mix+B18R Protein 3 0.6 μg mRNA mix None 0iPSC Colonies from BJ Fibroblasts Redifferentiated into all Three GermLayers.

Multiple colonies were manually picked from the first RNAiMAX™transfection experiment (Table 5) and plated onto new Nuff feeder layersin iPSC media with 100 ng/ml hFGF2. These colonies were passaged between5 and 10 times before some were frozen while other were tested forexpression of stem cells markers like OCT4, NANOG, SOX2 (FIG. 16). OtheriPSC clones were tested for their ability to differentiate into thethree germ layers as would be expected if these iPSC colonies are trulypluripotent. Three different iPSC clones made from BJ fibroblaststransfected using RNAiMAX™ with the 3:1:1:1:1 molar ratio of RNaseIII-treated Ψ- and m⁵C-modified miRNAs encoding OCT4, SOX2, KLF4, LIN28and c-MYC at a dose of 1.2 micrograms/ml per day for 18 days werepassaged 7 times and tested in embryoid body spontaneous differentiationassays (Huangfu et al., 2008). After 8 days in suspension culturefollowed by 8 more days of attachment to gelatin coated plates, all 3clones differentiated into all three germ layers marked by endodermmarkers (AFP and SOX17), mesoderm markers (SMA and Desmin) and theectoderm neuronal marker class III beta-tubulin) (FIGS. 17A, B and C).With iPSC Clone 2, beating cardiac myocytes were generated in one of thewells.

iPSC Induction from BJ Fibroblasts Using TransIT™ Protocols

Similar iPSC reprogramming experiments performed using the TransIT™ mRNAtransfection reagent (Mirus Bio, Madison, Wis., USA) resulted in manymore iPSC colonies and the iPSC colonies appeared earlier than wereobtained using RNAiMAX™ transfection reagent (FIG. 18). For example, wewere able to see iPSC colonies forming in the wells transfected dailywith the 1.2-micrograms dose of the 3:1:1:1:1 molar ratio of RNaseIII-treated Ψ- and m⁵C-mRNAs encoding OCT4, SOX2, KLF4, LIN28 and c-MYCwells as early as transfection day 10.

TABLE 7 iPSC induction from BJ fibroblasts transfected with RNaseIII-treated Ψ-modified or Ψ- and m⁵C-modified mRNAs using TransIT ™.Number of Alkaline Number Phosphatase-Positive on Plate Treatment iPSCColonies on Day 15 1 Mock Transfected 0 2 1.2 μg of Ψ- and m⁵C-mRNA 3213 1.0 μg of Ψ- and m⁵C-mRNA 125 4 0.8 μg of Ψ- and m⁵C-mRNA 13 5 0.6 μgof Ψ- and m⁵C-mRNA 0 6 0.4 μg of Ψ- and m⁵C-mRNA 0 7 Mock Transfected 08 1.2 μg of Ψ-mRNA 49 9 1.0 μg of Ψ-mRNA 168 10 0.8 μg of Ψ-mRNA 8 110.6 μg of Ψ-mRNA 4 12 0.4 μg of Ψ-mRNA 0

Table 7 shows the number of iPSC colonies generated from human BJfibroblasts transfected daily with amounts of the 3:1:1:1:1 molar ratioof RNase III-treated Ψ- or Ψ- and m⁵C-modified mRNAs encoding iPSCinduction factors (OCT4, SOX2, KLF4, LIN28, cMYC), as indicated. After15 days, the cells were fixed and stained for alkaline phosphatase, astem cell marker.

Evaluation of iPSC Induction Using mRNAs Encoding Different Combinationsof Reprogramming Factors

Based on the finding that the TransIT™ reagent resulted in an impressiveincrease in the number iPSC colonies generated from BJ fibroblastscompared to using the RNAiMAX™ reagent, the TransIT™ reagent was thenused to evaluate different combinations of reprogramming factors.

We found that use of mRNAs encoding the four iPSC induction factors(OCT4, SOX2, KLF4, c-MYC) shown to generate iPSCs by Takahashi andYamanaka (2006) were sufficient to reprogram BJ fibroblasts to alkalinephosphatase-positive iPSC colonies by reprogramming day 18. In theseexperiments, induction of iPSC colonies using Ψ- and m⁵C-modified mRNAinduction factors encoding OCT4, SOX2, KLF4, c-MYC did not appear to bemore efficient than using only Ψ-modified mRNAs that encoded thoseproteins (FIG. 19).

Yu et al. (2007) showed that human somatic cells could be reprogrammedby overexpressing OCT4, SOX2, LIN28 and NANOG as reprogramming factorsusing lentiviral systems. Possibly higher amounts of the mRNAs or moretreatments with mRNAs encoding these factors could be successful.However, we did not observe any alkaline phosphatase-positive iPSCcolonies by reprogramming day 18 using the amounts of RNase III-treatedmRNA induction factors encoding only OCT4, SOX2, LIN28 and NANOGevaluated, whether those mRNAs were only Ψ-modified or both Ψ- andm⁵C-modified.

Nakagawa M et al. (2010) showed that L-MYC, a member of the MYC oncogenefamily with less oncogenic activity than c-MYC, could be used in placeof c-MYC for iPSC induction. We found that RNase III-treated Ψ-modifiedor Ψ- and m⁵C-modified mRNA encoding L-MYC was also able to be used inplace of c-MYC for iPSC reprogramming of BJ fibroblasts in variouscombinations of mRNA iPSC induction factors. However, efficiency of iPSCcolony generation using mRNAs encoding L-MYC was generally lower thanwhen using mRNAs encoding c-MYC (Table 8 and FIG. 19 and FIG. 20).

Nakagawa M et al. (2010) showed that human cells can be reprogrammed toa pluripotent state by using lentiviral systems to overexpress onlythree factors (OCT4, SOX2 and KLF4). We did not observe generation ofalkaline phosphatase-positive iPSC colonies by reprogramming day 18using the amounts of RNase III-treated mRNA induction factors encodingonly OCT4, SOX2 and KLF4 without c-MYC or L-MYC, whether those mRNAswere only Ψ-modified or both Ψ- and m⁵C-modified (Table 8 and FIG. 19).Possibly higher amounts of the mRNAs or more treatments with mRNAsencoding these factors could be successful.

When reprogramming factors were expressed in somatic cells usingepisomal vectors, others have found that introduction of expression of 6factors (OCT4, SOX2, KLF4, LIN28, c-MYC and NANOG) resulted in thehighest level of iPSC induction.

We found that RNase III-treated Ψ-modified or Ψ- and m⁵C-modified mRNAencoding all six factors, including NANOG, generally resulted in aslight increase in the number of alkaline phosphatase- (ALKP-) positivecolonies compared to OCT4, SOX2, KLF4, LIN28 and c-MYC without NANOG(Table 8 and FIG. 19). In this particular experiment, we also observedformation of iPSC colonies on day 10 when mRNA encoding NANOG wasincluded, compared to day 11 or day 12 when mRNA encoding NANOG was notincluded with the OCT4, SOX2, KLF4, LIN28 and c-MYC mRNAs.

TABLE 8 iPSC Induction of BJ Fibroblasts by different kinds and amountsof reprogramming mRNAs. No. of Alkaline Phosphatase- Total Amount,Identity and Positive Plate Modification of RNase III-treated ColoniesNumbering mRNAs Used for Transfection On Day 17 Plate 5 Untreated BJFibroblasts 0 (last 2 wells) Plate1 (A1.2) 1.2 μg Total Ψ-mRNA Encoding108 OCT, SOX2, KLf4, LIN28, cMYC Plate1 (A1.0) 1.0 μg Total Ψ-mRNAEncoding 278 OCT, SOX2, KLf4, LIN28, cMYC Plate1 (B1.2) 1.2 μg Total Ψ-& m⁵C-mRNA Encoding 85 OCT, SOX2, KLf4, LIN28, cMYC Plate1 (B1.0) 1.0 μgTotal Ψ- & m⁵C-mRNA Encoding 268 OCT, SOX2, KLf4, LIN28, cMYC Plate1(C1.2) 1.2 μg Total Ψ-mRNA Encoding 36 OCT, SOX2, KLf4, LIN28, L-MYCPlate1 (C1.0) 1.0 μg Total Ψ-mRNA Encoding 34 OCT, SOX2, KLf4, LIN28,L-MYC Plate2 (D1.2) 1.2 μg Total Ψ- & m⁵C-mRNA Encoding 171 OCT, SOX2,KLf4, LIN28, L-MYC Plate2 (D1.0) 1.0 μg Total Ψ- & m⁵C-mRNA Encoding 107OCT, SOX2, KLf4, LIN28, L-MYC Plate2 (E1.2) 1.2 μg Total Ψ-mRNA Encoding28 OCT, SOX2, KLf4, cMYC Plate2 (E1.0) 1.0 μg Total Ψ-mRNA Encoding 87OCT, SOX2, KLf4, cMYC Plate2 (F1.2) 1.2 μg Total Ψ- & m⁵C-mRNA Encoding207 OCT, SOX2, KLf4, cMYC Plate2 (F1.0) 1.0 μg Total Ψ- & m⁵C-mRNAEncoding 255 OCT, SOX2, KLf4, cMYC Plate3 (G1.2) 1.2 μg Total Ψ-mRNAEncoding OCT, 0 SOX2, KLf4, L-MYC Plate3 (G1.0) 1.0 μg Total Ψ-mRNAEncoding 3 OCT, SOX2, KLf4, L-MYC Plate3 (H1.2) 1.2 μg Total Ψ- &m⁵C-mRNA Encoding 44 OCT, SOX2, KLf4, L-MYC Plate3 (H1.0) 1.0 μg TotalΨ- & m⁵C-mRNA Encoding 17 OCT, SOX2, KLf4, L-MYC Plate3 (I1.2) 1.2 μgTotal Ψ-mRNA Encoding 0 OCT, SOX2, KLf4 Plate3 (I1.0) 1.0 μg TotalΨ-mRNA Encoding 0 OCT, SOX2, KLf4 Plate4 (J1.2) 1.2 μg Total Ψ- &m⁵C-mRNA Encoding 0 OCT, SOX2, KLf4 Plate4 (J1.0) 1.0 μg Total Ψ- &m⁵C-mRNA Encoding 0 OCT, SOX2, KLf4 Plate4 (K1.2) 1.2 μg Total Ψ-mRNAEncoding 97 OCT, SOX2, KLf4, LIN28, cMYC, NANOG Plate4 (K1.0) 1.0 μgTotal Ψ-mRNA Encoding 364 OCT, SOX2, KLf4, LIN28, cMYC, NANOG Plate4(L1.2) 1.2 μg Total Ψ- & m⁵C-mRNA Encoding 150 OCT, SOX2, KLf4, LIN28,cMYC, NANOG Plate4 (L1.0) 1.0 μg Total Ψ- & m⁵C-mRNA Encoding 303 OCT,SOX2, KLf4, LIN28, cMYC, NANOG Plate5 (M1.2) 1.2 μg Total Ψ-mRNAEncoding 0 OCT, SOX2, LIN28, NANOG Plate5 (M1.0) 1.0 μg Total Ψ-mRNAEncoding 0 OCT, SOX2, LIN28, NANOG Plate5 (N1.2) 1.2 μg Total Ψ- &m⁵C-mRNA Encoding 0 OCT, SOX2, LIN28, NANOG Plate5 (N1.0) 1.0 μg TotalΨ- & m⁵C-mRNA Encoding 0 OCT, SOX2, LIN28, NANOG

Example 13. Reprogramming BJ Fibroblasts to iPS Cells Using RNaseIII-Treated Cap1 Poly-A-Tailed y-Modified mRNAs Encoding OCT4, SOX2,KLF4, LIN28, NANOG and cMYC Reprogramming Factors Materials and Methodsfor Example 13.

mRNA Reprogramming Factors

Cap1 5′-capped ψ-modified mRNAs encoding OCT4, SOX2, KLF4, LIN28, NANOGand cMYC with an approximately 150-base poly(A) tail (with tail lengthverified by denaturing agarose gel electrophoresis) were prepared andthen mixed in a 3:1:1:1:1:1 molar as described above. The RNase IIItreatment to remove dsRNA was performed using the in vitro-transcribedRNA prior to capping and tailing in the presence of 1 mM magnesiumacetate.

Brief Description of the Reprogramming Method

BJ fibroblasts cells (ATCC) were plated onto irradiated feedercells-human NuFF cells (newborn foreskin fibroblasts; Globalstem) inPLURITON™ medium plus supplement (Stemgent) with penicillin/streptomycin(pen/strep) antibiotics, and transfected daily for 18 days with 800 ngof the 3:1:1:1:1:1 mRNA mix TransIT™ mRNA transfection reagent (2microliters per microgram of RNA; Mirus Bio). The cell media was changeddaily, 1 hour prior to transfection, with SCRIPTGUARD™ RNase inhibitor(0.4 U/ml; CELLSCRIPT) added to the media prior to transfection. Thecells in each culture well were split 1-to-3 and transferred into 3replicate wells on Day 8 after the 9th transfection.

Detailed Description of the Reprogramming Method

2.5×10⁵ passage 9, mitotically-inactivated NuFF cells (GlobalStem) wereplated on gelatin-coated, 6-well plates in NuFF media (DMEM [LifeTechnologies], 10% fetal bovine serum (FBS, Thermo Fisher), 1× GLUTAMAX(Life Technologies) and 1× penicillin/streptomycin (Life Technologies).Twenty-four hours later, the media was removed and 104 BJ fibroblasts(ATCC) were plated per well on the NuFF feeder cells in fibroblastmedium, (EMEM [ATCC] supplemented with 10% FBS and 1× pen/strep).

PLURITON™ reprogramming medium (Stemgent), with freshly added 1×PLURITON™ supplement and 1× pen/strep, was changed daily one hour priorto mRNA transfections. SCRIPTGUARD™ RNase inhibitor was added to thePLURITON™ reprogramming media (and to the NuFF-conditioned PLURITON™reprogramming media described below) to a final concentration of 0.4U/ml. The medium was then added to cells daily, 1 hour or less beforethe transfections.

Cells were transfected on 18 consecutive days using the TransIT®-mRNAtransfection kit (Mirus Bio). 800 ng reprogramming mRNA mix was dilutedin 250 microliters Opti-MEM I (Life Technologies) and 1.6 microliters ofTransIT BOOST™ was added, reaction components were mixed, then 1.6microliters of the TransIT transfection reagent was added and thereaction components were mixed. After 2-5 minutes incubation at roomtemperature, the transfection mix was applied drop-wise to cells. Cellswere then incubated at 37° C. in 5% CO₂ overnight. After 6 dailytransfections in PLURITON™ medium, the medium was changed toNuFF-Conditioned PLURITON™ reprogramming media. his PLURITON™-basedmedium was previously incubated on NuFF feeder cells for 24 hours, wascollected and stored frozen until used. When needed, the conditionedmedium was thawed, filtered and PLURITON™ supplement and antibioticswere added fresh, daily. After the last of the 18 daily transfections,the cells were live stained with an antibody to TRA-1-60, to confirmiPSC colony production.

In Vivo Immunostaining Methods

A sterile, TRA-1-60 antibody (StainAlive™ DyLight™ 488 anti-humanTRA-1-60 antibody; Stemgent) was diluted 1:100 in PLURITON™ medium. Onday 18 of the reprogramming protocol, the medium was removed and thecells were incubated in TRA-1-60-containing media for 30 minutes at 37°C. with 5% CO₂. The cells were then washed twice with PLURITON™ mediumto remove the unbound antibody and the cells were maintained in freshreprogramming medium during immunofluorescent imaging. This antibodyallows live cell staining, instead of fixing the cells and sacrificingthem for the imaging. Based on morphology and TRA-1-60 antibodystaining, hundreds of iPSC colonies were generated per well of BJfibroblast cells transfected with pseudouridine-modified mRNA that hadbeen treated with RNase III, but only 2 iPSC colonies were seen with thedual-modified pseudouridine- and 5-methylcytidine-containing mRNA thathad been treated with RNase III. After iPSC cell colonies were confirmedby morphology and TRA-1-60 staining, on day 19 they were picked andplated on fresh NuFF feeder cells in NuFF-conditioned medium.

Picking of Reprogrammed iPSC Colonies

Colonies of iPSCs were manually picked from reprogramming plates andexpanded for further characterization. In manual picking, colonies weredissected with a pipette, physically removed from the reprogrammingplate, and the pieces were re-seeded onto fresh NuFF feeder cell plateswith 10 uM Y27632 ROCK inhibitor (Stemgent) in the NuFF-conditionedmedium. The cells were expanded and split when 60-70% confluent withcollagenase IV as described below.

Maintaining iPSCs

Induced pluripotent stem cell cultures were expanded and maintainedeither on feeder-dependent or feeder-free, MATRIGEL® 6-well plates. Infeeder-dependent culturing of iPSCs, the cells were maintained on eitherirradiated human neonatal fibroblasts (GlobalStem) or irradiatedembryonic mouse fibroblasts (R&D Systems) seeded at 2.5×10⁵ cells perwell. iPSC colonies on feeder plates were kept in iPSC maintenancemedium, DMEM/F12 supplemented with 20% Knockout Serum Replacement, 1 mML-Glutamine, 0.1 mM non-essential amino acids solution, 10 ng/ml basichuman recombinant FGF (all from Invitrogen) with penicillin-streptomycinantibiotics. The medium was changed daily. Cultures were split when thecell population grew to about 60% to 70% confluency using collagenase IVas described below.

In feeder-free culturing of iPSCs, colonies were maintained on 6-welltissue culture plates coated with 83 ng per well of MATRIGEL™ (BDBiosciences). Colonies on MATRIGEL™ plates were kept in mTESR (STEMCELLTechnologies) media that was changed daily. Cultures were split when thecell population grew to about 60% to 70% confluency using dispase asdescribed below.

Splitting iPSCs

For cultures maintained on feeders, the day before splitting, 0.1%gelatin coated plates were seeded with feeder cells, either irradiatedhuman neonatal fibroblasts (GlobalStem) or irradiated embryonic mousefibroblasts (R&D Systems) at 2.5×10⁵ cells per well. Cells were washedonce with 1× phosphate-buffered saline solution (PBS), and 1 ml of 1mg/ml collagenase type IV solution in DMEM/F12 (Invitrogen) was applied.iPSC colonies were incubated in collagenase IV at 37° C. and 5% CO₂ for8 to 10 minutes until the edges of the colonies began to lift up.Colonies were gently washed 3 times with 2 to 3 mls of DMEM/F12, andremoved and broken up in iPSC maintenance medium, DMEM/F12 supplementedwith 20% Knockout Serum Replacement, 1 mM L-Glutamine, 0.1 mMnon-essential amino acids solution, 10 ng/ml basic human recombinant FGF(all from Invitrogen) with penicillin-streptomycin antibiotics, involumes to reach the appropriate split ratios. Split cultures wereplated on fresh plates pre-seeded with feeders in iPSC maintenancemedium.

For feeder-free maintenance of iPSC cultures, 6 well tissue cultureplates were coated with 83 ng per well of MATRIGEL (BD Biosciences) atroom temperature at least one hour prior to use. In passaging iPSCcultures, medium was removed and replaced with 1 ml of a 1 mg/ml dispasesolution in DMEM/F12(STEMCELL Technologies). Cultures were incubated at37° C. and 5% CO₂ for 8 to 10 minutes until the edges of the coloniesbegan to lift up. iPSC colonies were gently washed 3 times with 2 to 3mls of DMEM/F12, and removed and broken up in fresh media, mTESR™(STEMCELL Technologies) before plating on a new MATRIGEL plate.

On day 28, after 10 days of culture, after picking colonies on day 19and one subsequent collagenase passaging of the cells, some of the cellswere fixed and immunostained.

Methods for Immunostaining of iPSCs

iPSC colonies were washed twice in 1× phosphate-buffered solution (PBS)and fixed in 4% paraformaldehyde in PBS at room temperature for half anhour. After 3 washes in 1×PBS, cells were washed 3 times in wash buffer,(PBS with 0.1% Triton-X100), and blocked for one hour at roomtemperature in blocking solution, 0.1% triton-X100, 1% BSA, 2% FBS inPBS. Primary antibodies were diluted 1:500 in blocking solution andapplied to cells overnight at 4° C. Cells were washed 6 times in washbuffer. Secondary antibodies were diluted 1:1,000 in blocking buffer,were applied for 2 hours at room temperature in the dark. After 6 washeswith wash buffer, cells were washed twice in 1×PBS before imaging.Images are shown in FIG. 13. FIG. 15, FIG. 16, FIG. 17, FIG. 22, FIG. 31and FIG. 26.

Primary Antibodies Used:

Oct4 Rabbit Antibody (Santa Cruz Biotechnology)

Tra-1-60 Mouse Antibody (Cell Signaling Technology)

Lin28 Mouse Antibody (Cell Signaling Technology)

NANOG Rabbit Antibody (Cell Signaling Technology)

SSEA4 Mouse Antibody (Cell Signaling Technology)

Secondary Antibodies Used:

Alexa Fluor® 488 Anti-Rabbit (Molecular Probes, Life Technologies)

Alexa Fluor® 555 Anti-Mouse (Molecular Probes, Life Technologies)

Differentiation into Cardiomyocytes

Some iPSC colonies were differentiated into cardiomyocytes as describedin EXAMPLE 11.

Results for Example 13.

By day 18, >>100 colonies of iPSC colonies were present in each of 3replicate wells of BJ fibroblasts that had been transfected with 18daily doses of 800 ng of the 3:1:1:1:1 molar mix of RNase III-treated,pseudouridine-modified mRNA encoding OCT4, SOX2, KLF4, LIN28, NANOG andcMYC. iPSC colonies were too numerous to count and some iPSC colonieswere already beginning to differentiate into other cell types by day 18.(100 iPSC colonies would represent an efficiency of ˜1% iPSC induction).

The iPSC colonies exhibited iPSC colony morphology (FIG. 21).

Live iPSC colonies stained positively for the stem cell marker Tra-1-60(FIG. 22).

One of the 3 wells was also treated with B18R protein from Transfection#10 to #18, but no benefit was seen in that this well had a similarnumber of iPSC colonies as the well that did not receive B18R protein.

Greater than 50 iPSC colonies were picked on Day 19. Some iPSC colonieswere collagenase-treated and transferred; the remaining colonies weretransferred to new feeder cells on day 21.

Of iPSC colonies that were cultured >90% survived and were cultured forgreater than 10 passages.

Some of the iPSC colonies were fixed and immunostained positively forstem cell markers on day 28, 10 days of iPSC culture after the lasttransfection (FIG. 23).

Some of the iPSC colonies were propagated and differentiated intobeating cardiomyocytes.

Example 14. Additional Experiments on Reprogramming of BJ Fibroblasts toiPSCs and Further Characterization of iPSC Colonies Materials andMethods for Example 14.

Brief Description of the Reprogramming Method

The iPSC reprogramming factors were composed of cap1 5′-cappedψ-modified mRNAs encoding OCT4, SOX2, KLF4, LIN28, NANOG and cMYC withan approximately 150-base poly(A) tail (with tail length verified bydenaturing agarose gel electrophoresis) were prepared and then mixed ina 3:1:1:1:1:1 molar as described above. The RNase III treatment toremove dsRNA was performed using the in vitro-transcribed RNA prior tocapping and tailing in the presence of 1 mM magnesium acetate. BJfibroblasts cells (ATCC) were reprogrammed using the iPSC reprogrammingfactors in a similar reprogramming method to that described in EXAMPLE13, except that the BJ fibroblasts were transfected daily for 18 dayswith one microgram (instead of 800 ng) of the 3:1:1:1:1 mRNA mixTransIT™ mRNA transfection reagent (2 microliters per microgram of RNA;Mirus Bio). Some BJ fibroblast cells in this EXAMPLE 14 were pretreatedwith B18R recombinant protein (eBioscience) prior to being treated withthe mRNA reprogramming factors, in which cases, the B18R proteinsolution was added to the reprogramming medium several hours beforeadding the mRNA reprogramming factors. Some of the iPSC colonies werealso transferred to an artificial extracellular (MATRIGEL™) matrix forpropagation in order to propagate the iPSCs in the absence of feedercells. The iPSCs propagated on MATRIGEL matrix were used for isolationand purification of mRNA from the iPSCs for gene expression analysis byqRT-PCR without also isolating contaminating feeder cell mRNA. Forfeeder-free culturing, iPSC colonies were maintained on 6-well platescoated with hES-qualified MATRIGEL™ matrix (BD Biosciences). TheMATRIGEL matrix was thawed on ice, diluted in DMEM/F12 media and plateswere coated for one hour at room temperature prior to use. iPSC colonieson MATRIGEL-coated plates were kept in mTESR medium (STEMCELLTechnologies) that was changed daily. Cultures were split when the cellpopulation grew to about 60% to 70% confluency as described below. Inpassaging iPSC cultures, medium was removed and replaced with 1 ml of a1 mg/ml dispase solution in DMEM/F12 medium (STEMCELL Technologies).Cultures were incubated at 37° C. and 5% CO₂ for 8 to 10 minutes untilthe edges of the colonies began to lift up. Colonies were gently washed3 times with 2 to 3 mls of DMEM/F12, and removed and broken up in freshmTESR medium (STEMCELL Technologies) before plating on a newMATRIGEL-coated plate. mTeSR was supplemented with 10 uM Y27632 ROCKinhibitor (Stemgent). Plates were incubated at 37° C. in 5% CO₂overnight. Cultures were maintained in mTESR for expansion and RNApurification for qRT-PCR experiments as described below.

Detailed Description of the Reprogramming Method for Example 14.

The BJ fibroblast cells were reprogrammed to iPSCs using the materials,methods and protocols presented below.

Materials—for Reprogramming of Human Fibroblasts to iPSCs.

-   -   PLURITON™ Reprogramming Media (Stemgent, Cat #00-0070)    -   DMEM, High glucose (GIBCO, Cat #11965-092, Life Technologies)    -   EMEM (ATCC, Cat #302003)    -   Defined fetal bovine serum (Hyclone Cat #SH30070.03, Thermo)    -   GLUTAMAX™-1 (GIBCO, Cat #35050-061, Life Technologies)    -   Penicillin 10000 IU/Streptomycin 10000 micrograms (200×) (MP        Biomedicals, Cat #1670249, Thermo)    -   OPTI-MEM® I Reduced Serum Medium 1× (Invitrogen, Cat #11058-021)    -   Neonatal Human Foreskin Fibroblasts (NuFF), P9, IRR (GlobalStem        Cat #GSC-3001G)—passage 9, irradiated    -   BJ Human Newborn Fibroblasts (ATCC, Cat #CRL-2522)    -   B18R Recombinant Protein Carrier-Free (eBioscience Cat        #34-8185-85)    -   Recombinant Human Fibroblast Growth Factor-basic (FGFb) (AA        10-155) (GIBCO, Cat #PHG0023, Life Technologies)    -   TransIT®-mRNA Transfection Kit (Mirus Bio, Cat #2256)    -   UltraPure water with 0.1% Gelatin (Millipore, Cat #ES-006-B)    -   0.025% Trypsin, 0.02% EDTA for Primary Cells (GIBCO, Cat        #R-001-100, Life Technologies)    -   Trypsin Neutralizer Solution (GIBCO, Cat #R-002-100, Life        Technologies)

Media Composition

-   -   NuFF Culture Medium—DMEM high glucose, 10% defined FBS, 1×        GlutaMAX-1, 1× Pen/Strep    -   BJ Fibroblast Medium—EMEM, 10% defined FBS, 1× Pen/Strep    -   PLURITON™ Reprogramming Medium—Pluriton Medium, 1× Pluriton        Supplement, 1× Pen/Strep    -   NuFF Conditioned, PLURITON™ Reprogramming Medium—25 ml Pluriton        Reprogramming Medium (above) collected after 24 hours on 4×10⁶        NuFFs cells, collected daily, pooled, filter sterilized, and        stored in frozen aliquots.

Solution Composition

-   -   FGFb—dilute to 4 micrograms/ml and 50 micrograms/ml working        stocks in PBS with 0.1% BSA    -   Collagenase—make up 1 mg/ml in DMEM/F12 media

Preparation

-   -   Thaw Pluriton Supplement at 4° C. and aliquot and freeze at −70°        C.    -   Thaw Pluriton Media at 4° C. for 2 days    -   Thaw at 4C and aliquot B18R protein, store at −70° C.    -   Coat flasks with gelatin 4+ hours before needed for NuFF cells    -   Plate 4×10⁶ NuFFs per T75 flask in 25 ml media—for conditioned        media (takes 7 days)    -   Coat 6-well plates with gelatin for 4+ hours before needed for        NuFF cells    -   Plate NuFFs in 6 well dishes for reprogramming (day −2)    -   Plate BJ fibroblasts 10′ cells per well of 6-well dish    -   Transcribe, cap, tail and quantify mRNA    -   Mix KLMOS mRNA to 1:1:1:3:1 molar ratio in sterile water    -   Make Pluriton media fresh daily by adding supplement and        Pen/Strep to 1×

Generation of NuFF-Conditioned Pluriton Medium (Start on Day −2)

-   -   1. Add 8 ml 0.1% gelatin solution to a T75 tissue culture flask.    -   2. Incubate the flask at least 4 hours at 37° C. and 5% CO₂.    -   3. Plate inactivated Newborn Human Foreskin Fibroblasts (NuFFs)        at a density of 4×10⁶ cells in 25 ml of NuFF Culture Medium in a        T75 flask.    -   4. Incubate the NuFF cells overnight at 37° C. and 5% CO₂.    -   5. The following day, aspirate the NuFF Culture Medium from the        flask and discard.    -   6. Add 10 ml of PBS to wash. Aspirate the PBS and discard.    -   7. Add 25 ml of Pluriton Medium supplemented with 25 microliters        of 4 micrograms/ml FGF-basic Solution and 125 microliters of        200× Penicillin/Streptomycin to the NuFFs in the T75 flask.    -   8. Incubate the cells and medium overnight at 37° C. and 5% CO₂.    -   9. After 24 hours, collect the NuFF-Conditioned Medium and store        at −20° C.    -   10. Add 25 ml of fresh Pluriton Medium supplemented with 25        microliters of FGFb Solution and 125 microliters of        Penicillin/Streptomycin to the NuFFs in the T75 flask.    -   11. Incubate overnight at 37° C. and 5% CO₂.    -   12. Repeat steps 7 through 9 daily for five additional days.        Pool in orange capped sterile bottle and keep at −20C until        final collection. Note: Six days of medium collection will yield        a total of ˜150 ml of NuFF-Conditioned Pluriton Medium.    -   13. Thaw all frozen aliquots of NuFF-Conditioned Pluriton Medium        at 4° C.    -   14. Pool aliquots and filter-sterilize using a 0.22 μm pore        size, low protein-binding filter.    -   15. Aliquot 20-40 ml of the filtered NuFF-Conditioned Pluriton        Medium into 50 ml conical tubes.    -   16. Store aliquots at −20° C. until needed on Days 6 to 20 of        reprogramming.

Prior to use of NuFF-Conditioned Pluriton Medium:

-   -   1. Thaw one aliquot of NuFF-Conditioned Pluriton Medium and one        aliquot of Pluriton Supplement 2500× at 4° C.    -   2. Just prior to use, add 4 microliters of the Pluriton        Supplement 2500× to 10 ml of equilibrated NuFF-Conditioned        Pluriton Medium.

Reprogramming Timeline

-   -   Day minus 2 Gelatin Coat plates—incubate 4 hours at 37° C.        -   Plate NuFF cells.        -   (Also plate NuFF cells to make conditioned medium.)    -   Day minus 1 Plate BJ cells on NuFF cells.        -   (Change medium on NuFF flasks for conditioned medium.)    -   Day 0-5 Change medium to fresh Pluriton Reprogramming Medium.        -   Transfect Cells and Collect conditioned medium from flasks            to use from Day 6-17.)    -   Day 6-17 Change media to fresh NuFF conditioned Pluriton        Reprogramming Medium.        -   Transfect Cells.    -   Day 18 Examine Cells and identify colonies.        -   Gelatin coat fresh plates and plate NuFF feeder cells.    -   Day 19+ Pick colonies and transfer onto fresh NuFF feeder cells.        -   Change media to iPSC media.    -   ˜Day 18-22 Cells can be fixed, stained, collagenased to new        plates or MEF or NuFF feeder cells, plated on MATRIGEL™ and the        media can be changed to a number of ES or iPS cell media.        Step-by-Step Protocol Used for Reprogramming of Human        Fibroblasts to iPSCs:

Plate Human NuFF Feeder Cells (Day −2)

-   -   1. Add 1 ml of 0.1% gelatin in 6 wells of a 6-well tissue        culture plate.    -   2. Incubate the plate at least 4 hours at 37° C. and 5% CO2.    -   3. Thaw one vial (4-5×10⁶ cells) of mitotically inactivated        human newborn foreskin fibroblasts (NuFFs) and plate at a        density of 2.5 to 5×10⁵ cells per well in NuFF Culture Medium in        the 6 wells of the 6-well plate coated with gelatin.    -   4. Incubate the cells overnight at 37° C. and 5% CO2.        Plate the BJ Fibroblasts on the feeder layer (day −1)    -   1. Aspirate the NuFF culture medium from the cells and discard.    -   2. Plate BJ fibroblasts at 1×10¹ cells per well in BJ media on        top of the NuFF cells.    -   3. Incubate cells overnight at 37° C. and 5% CO2.

Reprogram Cells (day 0)

Add B18R protein to the wells of cells to be pre-treated with thisprotein inhibitor.

-   -   1. Aspirate the BJ medium from the target cells and add 2 ml per        well of Pluriton Complete with or without B18R protein to a        final concentration of 200 ng/ml.    -   2. Incubate the plate for a minimum of 4 hours at 37° C. and 5%        CO₂ if using B18R protein. NOTE: If not using B18R protein,        incubate the plate at 37° C. and 5% CO₂ for 1 hour before the        first transfection.        Prepare mRNA Transfection Complex Comprising the mRNA        Reprogramming Mix    -   1. Thaw mRNA reprogramming mix on ice.    -   2. Add 250 microliters of OPTI-MEM to a sterile 1.5-ml        microcentrifuge tube.    -   3. Add 8-12 microliters of 100 ng/microliter mRNA reprogramming        Premix to the OPTI-MEM and pipet to mix.    -   4. Add 2 microliters of TransIT Boost Reagent per 1 microgram of        mRNA used. Pipet up and down to mix.    -   5. Add 2 microliters of TransIT mRNA Transfection Reagent per 1        microgram of mRNA used. Pipet to mix.    -   6. Incubate at RT 2-5 minutes and add drop wise to cells.    -   7. Gently rock the 6-well plate from side-to-side and        front-to-back to distribute the mRNA Transfection Complex across        the well.    -   8. Incubate the plate for −23 hours at 37° C. and 5% C02.    -   Notes: The transfection complex comprising the mRNA        reprogramming mix must be mixed well after each addition to        cells to ensure the best transfection efficiency. Only a few        reactions should be prepared at a time, so that the mRNA        transfection reagent can be added quickly after the TransIT®        Boost reagent.

Reprogram Cells (for Day 1 Through Day 5)

-   -   1. Equilibrate Pluriton medium and make Complete with P/S and        Supplement (and B18R protein when included in treatment).    -   2. Aspirate the culture medium and discard.    -   3. Add 2 ml of Pluriton mRNA Reprogramming Medium to each well        (including with B18R protein when used in the treatment).    -   4. Incubate at 37° C. and 5% CO2 for 1 hour.    -   5. Prepare mRNA Transfection Complex as described for day 0.    -   6. Transfect cells as described for day 0.    -   7. Incubate the plate O/N at 37° C. and 5% CO₂.    -   8. Repeat steps 1 through 8 four additional times (day 2 through        day 5).

Reprogram Cells on Each of Days 6 Through 17, Each Time Changing theMedium to NuFF Conditioned Medium and Continuing Transfections.)

-   -   1. Thaw NuFF Conditioned Pluriton media and supplement and B18R        protein    -   2. Equilibrate NuFF Pluriton Media and make Complete with P/S        and Supplement (and B18R protein when used)    -   3. Aspirate the culture medium containing the mRNA Transfection        Complex.    -   4. Add 2 ml of conditioned mRNA Reprogramming Medium (with or        without B18R protein) to each well.    -   5. Incubate at 37° C. and 5% CO2 for 1 hour.    -   6. Prepare mRNA Transfection Complex as described for day 0.    -   7. Transfect cells as described for day 0.    -   8. Incubate the plate O/N at 37° C. and 5% CO2.    -   9. Repeat steps 1 through 8 twelve additional times (day 6        through day 17).

Identification of Primary iPS Cell Colonies (Day 18 Through Day 20)

-   -   1. After transfections are completed, incubate for 1 to 3 days        to allow colonies to expand.    -   2. Replace medium daily with 2 ml per well of NuFF-Conditioned        Pluriton Medium with Pluriton Supplement 2500× but without B18R        protein.    -   3. Prior to manual isolation, primary iPS cell colonies can be        identified using sterile, live-staining antibodies, such as        StainAlive™ DyLight™ 488 Mouse anti-Human without harm to the        cells.    -   4. Cells can be next be fixed and stained for alkaline        phosphatase activity, fixed and stained with antibodies or kept        alive and picked or collagenase transferred to fresh fibroblast        feeder layer-coated plates.

APPENDIX B

Passaging Cells in mRNA Reprogramming Medium

-   Note: Cells in the most confluent wells may be passaged on    approximately day 6 or day 7 to allow for further proliferation and    colony formation.    -   Cells should be passaged after a 4 hour transfection, thereby        replacing the daily medium change. Passaging, if needed, should        take place after day 6 or day 7. A new plate containing NuFF        feeder cells at 2.5×10⁵ cells per well should be plated the day        prior to passaging, as done on Day Minus 2.    -   1. Warm Trypsin/EDTA and Trypsin Neutralizer in a 37° C.        waterbath.    -   2. Add 1 ml of PBS per well of cells to be passaged. Aspirate        the PBS wash.    -   3. Add 0.5 ml of Trypsin/EDTA to the well. Gently rock the plate        to evenly distribute the enzyme across the well.    -   4. Incubate the cells for 5 minutes at 37° C. and 5% CO₂.    -   5. Remove plate from the incubator and gently tap the side of        the well to assist the dissociation and release the cells from        the culture surface.    -   6. Add 0.5 ml of Trypsin Neutralizer to the well.    -   7. Gently pipet the cells in the well three times with a 1 ml        pipet tip.    -   8. Collect the cells and transfer to a 15 ml conical tube.    -   9. Add 1 ml of Pluriton Medium to the well to collect any        remaining cells.    -   10. Transfer the additional 1 ml of cells to the cell suspension        in the 15 ml conical tube.    -   11. Centrifuge for 5 minutes at 200× g.    -   12. Aspirate the supernatant and resuspend the pellet in 1 ml of        warm Pluriton Medium.    -   13. Aspirate the NuFF Culture Medium from the wells of a        prepared NuFF feeder plate.    -   14. Add 1 ml of PBS per well to rinse. Aspirate the PBS.    -   15. Add 2 ml of Pluriton mRNA Reprogramming Medium with B18R        protein and Y27632 to each well.        -   Note: B18R protein should be added to a final concentration            of 200 ng/ml and Y27632 ROCK inhibitor) should be added to a            final concentration of 10 μM.    -   16. Dispense the resuspended cells to the prepared wells of the        NuFF feeder plate.        -   Note: A 1:6 split ratio is recommended, but can be varied            depending on the confluency of the well and the            proliferation rate of the cells. One to 6 wells of cells can            be replated, however it is important to choose a number of            wells plated to continue reprogramming comparable with the            amount of mRNA available for the remainder of the            reprogramming experiment.    -   17. Incubate the cells overnight at 37° C. and 5% CO₂.

Materials for iPS Cell Growth, Isolation, Maintenance or Confirmation.

-   -   StainAlive DyLight 488 Mouse anti-Human Tra-1-60 Antibody        (Stemgent, Cat #09-0068)    -   Y27632 Rock I Inhibitor (Stemgent, Cat #04-0012)    -   10× PBS without calcium or magnesium(Lonza Biowhittaker, Cat        #17-517Q, Thermo)    -   Collagenase Type IV, 250 U/mg (GIBCO, Cat #17104-019)    -   iMEF—irradiated mouse embryonic fibroblasts (R&D Systems Cat        #PSC001)    -   BD MATRIGEL™ hESC-qualified Matrix (BD Cat #354277, Thermo)    -   mTeSR® 1 Medium Kit—Basal Medium plus 5× Supplement (STEMCELL        Technologies, Cat #05850)    -   Dispase 5 mg/ml (STEMCELL Technologies, Cat #07913)    -   Synth-a-Freeze, cell freezing media, (GIBCO, Cat #A12542-01,        Life Technologies)    -   DMEM/F12 (1:1) Media (GIBCO, Cat #11330, Life Technologies)    -   KNOCKOUT™ SR Serum Replacement for ES cells (GIBCO, Cat #10828,        Life Technologies)    -   MEM Non-Essential Amino Acids Solution NEAA (100×) (GIBCO, Cat        #11140, Life Technologies)    -   Beta-mercaptoethanol (Sigma, Cat #63689)    -   Alkaline Phosphatase Staining Kit II (Stemgent, Cat #00-0055)    -   Bovine Serum Albumin (for FGFb)    -   Paraformaldehyde 95% (Sigma, Cat #158127)    -   Antibodies, wash buffers, etc    -   iPS Cell Medium—DMEM/F12, 20% Knockout SR, 10 ng/ml FGFb, 1×        non-essential amino acids, 1× Pen/Strep, 0.1 mM        beta-Mercaptoethanol (bME), 1× GLUTAMAX™    -   mTeSR 1 Medium—mTeSR 1 plus 1× Supplement        Materials and Methods for Characterizing iPSC Colonies Generated        Using the Methods.

Immunostaining materials and methods were identical to those used inEXAMPLE 13, except that two additional antibodies—the TRA-1-81 MouseAntibody (Cell Signaling Technology) and DNMT 3B Rabbit Antibody (CellSignaling Technology)—were also used.

Q-PCR Assays of Gene Expression Levels in iPSCs Generated Using theMethods Compared to Expression Levels in BJ Fibroblast Somatic Cellsfrom which the iPSCs were Generated

In order to determine if genes that are known to be up-regulated inembryonic stem cells or iPS cells generated using other methods werealso up-regulated in the iPSCs generated using mRNA iPSC reprogrammingfactors according to the methods described herein, qRT-PCR was performedon total cellular RNA isolated from generated iPSC colonies and from BJfibroblasts.

Thus, total cellular RNA was isolated from BJ fibroblasts and from iPScell colonies grown on an artificial extracellular matrix (MATRIGEL™matrix by BD Bioscience) to minimize fibroblast contamination. The iPSCsused were obtained iPSC “clonal” colonies that had been picked andpassaged 5 times during a period of one month after the last day oftransfection with the mRNA reprogramming factors. An entire well ofthese “clonal” colonies was lysed and pooled for the RNA preparation.cDNA was synthesized by reverse transcription of 1 microgram of thetotal cellular RNA from BJ fibroblasts and from the clonal colonies ofiPSCs, respectively, using oligo d(T)₂₀VN primers. Then, real-time PCR(qPCR) was performed on the cDNAs using the SsoFAST™ EvaGreen PCRSupermix (BioRad) and PCR primers (designed based on information inAssen, 2008) to analyze the relative mRNA expression levels encoding thefollowing proteins:

GAPDH—a housekeeping gene, comparable in expression in both cell types.

NANOG—Nanog homeobox—involved in cell differentiation, proliferation,embryo development, somatic stem-cell maintenance and more.

OCT4—(POU5F1) POU class 5 homeobox 1—plays a role in embryonicdevelopment especially during early embryogenesis and it is necessaryfor ES cell pluripotency.

CRIPTO—(TDGF1) Teratocarcinoma-derived growth factor 1—anextra-cellular, membrane-bound signaling protein that plays an essentialrole in embryonic development and tumor growth.

GBX2—Gastrulation brain homeobox 2—a DNA binding transcription factorinvolved in a series of developmental processes.

GDF3—Growth Differentiation Factor 3—a member of the bone morphogeneticprotein (BMP) family and the TGF-beta superfamily. The members regulatecell growth and differentiation in both embryonic and adult tissues.

REX1—(REXO1) RNA exonuclease 1 homolog—involved in proliferation anddifferentiation

cMYC—a multifunctional, nuclear phosphoprotein acting as a transcriptionfactor that plays a role in cell cycle progression, apoptosis, andcellular transformation.

The cDNA samples were PCR-amplified in triplicate and the qPCR resultsobtained using the values were averaged and the data were expressed ascycle threshold or CT values. The CT value is the PCR cycle number atwhich the reporter fluorescence is greater than the threshold andproduces the first clearly detectable increase in fluorescence overbackground or baseline variability. This is the most accurate method ofcomparing expression levels by PCR before there is a plateau in productformation.

Embryoid Body Spontaneous Differentiation of iPSCs

The same iPSC colony line that was analyzed by qPCR was used in theembryoid body spontaneous differentiation protocol as described inEXAMPLE 12 in order to analyze the ability of the cells to differentiateinto cells representing all three germ layers. Briefly, a colony waspicked and expanded for 17 passages, then frozen down for a week, thenbrought up and passaged 4 more times. Large colonies were allowed toform, were detached from the MATRIGEL™ matrix surface with dispase, andwere kept in suspension culture for 8 days in iPS media with no FGFb toallow embryoid body formation. As described previously, the embryoidbodies were then plated on gelatin coated plates and allowed to attachand spontaneously differentiate in iPS media without FGFb for anadditional 7 days. The cells were then fixed and incubated withantibodies for various markers as previously described.Immunofluorescence was performed and the cells were imaged.

Results for Example 14.

By Day 10, there was a dramatic morphology change in the wells from longthin fibroblast morphology to smaller, rounder epithelial cellmorphology (FIG. 24). Based on the final results of this and otherreprogramming experiments with the mRNA reprogramming factors used, thismorphology change appears to be a reproducible sign that reprogrammingof the BJ fibroblasts to iPSC colonies will be successful.

iPSC colonies were first detected on Day 16 based on visual inspection(e.g., FIG. 25).

The iPSC colony counts in the wells on Day 18 were less impressive thanin EXAMPLE 13. Without being bound by theory, we believe that we damagedthe cells when we attempted splitting iPSC colonies using trypsin on Day10.

In this experiment, the presence of B18R protein, there were about 10times more iPSC colonies generated on Day 18 from the RNase III-treatedmRNA reprogramming factors that contained only pseudouridine than weregenerated by the same mRNA reprogramming factors that contained bothpseudouridine and 5-methylcytidine modifications.

iPSC colonies were picked from a well containing cells that werereprogrammed in the absence of B18R protein using 18 daily doses of 800ng of RNase III-treated mRNA reprogramming factors that contained onlypseudouridine modification, and were passaged and maintained inlong-term culture.

iPSC colonies were also picked from the well generated by the sametreatment regime but with B18R protein. These iPS cells were maintainedin culture for 2 months. No differences in morphology or propagationcharacteristics were observed between the iPSCs generated with orwithout B18R protein.

Some iPSC colonies were collagenase split and transferred to feedercells on Day 21.

Some iPSC colonies were fixed and immunostained on Day 46 (afterapproximately one month of iPSC culture after the last transfection(FIG. 26).

Some iPSC colonies were passaged on MATRIGEL matrix in the absence offeeder cells; after five passages and about one month in culture, RNAwas isolated from some of these iPSC colonies and for gene expressionanalysis by qPCR, as described. Examples of qPCR results are provided inFIG. 34 through FIG. 37.

Results of Gene Expression of iPSC Colonies Versus BJ Fibroblasts byqPCR

GAPDH primers were used to show that the amount of input cDNA andtherefore the input starting RNA amounts were equivalent. As shown inFIG. 34, both BJ fibroblasts and iPSC colonies expressed a large, almostequivilant amount of GAPDH, so CTs shown in FIG. 34 were not normalized.

Unlike the similar levels of GAPDH, the expression of every pluripotencyfactor (FIG. 35 to FIG. 37) was higher in the iPS cells than in the BJfibroblasts.

CRIPTO is a dramatic example of the change in expression levels. Theaverage cycle threshold for RNA encoding CRIPTO in BJ fibroblasts wasapproximately 30 cycles, whereas the average CT value for RNA encodingCRIPTO in iPSC colonies derived from the BJ fibroblasts wasapproximately 21 (FIG. 35); this 9-cycle difference represents a588-fold increase in CRIPTO expression. A CT of 20 cycles also indicatesthe large abundance of this message in the iPS cell RNA.

Summary of Expression Differences for all qRT-PCR Primer Pairs Tested

Protein encoded by RNA GAPDH NANOG OCT4 CRIPTO GBX2 GDF3 REX1 cMyc CT CTCT CT CT CT CT CT BJ Cells 18.5 30.4 29.6 30.1 32 ND ND 26.2 iPS Cells18.7 22.9 20.7 20.9 27.4 25.8 23.1 25.5 Delta CT 0.2 7.5 8.9 9.2 4.6 NDND 0.7 Fold Difference 1.15 181 478 588 24.25 ND ND 1.62

As would be expected for true iPSCs, all of the above markers except forthe housekeeping gene GAPDH were expressed at much higher levels in iPSCcolonies than in BJ fibroblasts. The similar CT values for GAPDH in bothtypes of cells, shows that equal amounts of RNA were compared. The folddifference was too great to be determined for BJ fibroblast genes withnondetectable (ND) levels of expression.

Pluripotency Demonstrated by Ability of iPSCs to SpontaneouslyDifferentiate into Embryoid Bodies Containing Cells of all Three GermLayers.

As shown in FIG. 27, the iPSCs induced by RNase III-treated [with 1 mMMg(OAc)₂], cap1 5′-capped, 150-base poly(A)-tailed, ψ-modified mRNAsencoding a 3:1:1:1:1:1 mixture of OCT4, SOX2, KLF4, LIN28, NANOG andcMYC and subjected to the embryoid body spontaneous differentiationprotocol stained positively for markers representing all 3 germ layersof cells, demonstrating the pluripotency of the cells. Thus, cells werefound that expressed the ectoderm markers, neuronal class III class IIIbeta-tubulin (TUJ1), Glial Fibrillary Acidic Protein (GFAP) andneurofilament-light (NF-L), the mesoderm markers, alpha-smooth muscleactin (SMA) and desmin, and the endoderm markers, transcription factorSOX17 and alpha-fetoprotein (AFP).

Example 15. Evaluations of HPLC Versus the RNase III Treatment Methodfor Preparing Reprogramming Factors Comprising Pseudouridine-ModifiedssRNA Encoding iPS Cell Induction Factors for Reprogramming BJFibroblasts to iPS Cells Materials and Methods for Example 15.

In one experiment, iPSC reprogramming factors composed of cap1 5′-cappedψ-modified mRNAs encoding OCT4, SOX2, KLF4, LIN28 and cMYC(T58), eachwith an approximately 150-base poly(A) tail (with tail length verifiedby denaturing agarose gel electrophoresis) were prepared as previouslydescribed, but without doing the RNase III treatment. The cap1,poly(A)-tailed mRNAs were each then split into 3 portions. One-third ofeach mRNA was purified by RNARx LLC (Wayne, Pa.) using HPLC as described(Karikó et al., 2011). One third of each mRNA was left unpurified andone third of each mRNA was treated with RNAse III using an RNase IIItreatment method with 1 mM Mg(OAc)₂ and cleaned up using the RNA QuickCleanup method described herein; (note, this time, the RNase IIItreatment was performed after capping and tailing had been done ratherthan after the in vitro transcriptions). Then, all 5 of the mRNAreprogramming factors from each portion (i.e., either all that had beenHPLC-purified, all that were unpurified, or all that were RNaseIII-treated) were mixed to a 3:1:1:1:1 molar ratio of ψ-modified mRNAsencoding, respectively, OCT4, SOX2, KLF4, LIN28 and cMYC(T58) to make anHPLC-purified mRNA reprogramming mix, and untreated mRNA reprogrammingmix, and an RNase III-treated reprogramming mix. Then, 1.2 micrograms ofeach mRNA reprogramming mix was used for reprogramming ten thousandcells per well of BJ fibroblasts (plated on NuFFs) to iPSCs, essentiallyas described in EXAMPLE 14, with additional experimental variables shownin the table of iPSC reprogramming results.

Spontaneous Differentiation of iPSCs into 3 Germ Layers

Selected iPSC colonies were picked and used in the embryoid bodyspontaneous differentiation protocol as describe in EXAMPLE 14 in orderto evaluate their pluripotency.

Results for Example 15.

iPSC colonies were detected by day 13 in wells of BJ fibroblaststransfected with the RNase III-treated mRNA reprogramming mix or withthe HPLC-purified mRNA reprogramming mix. All of the cells in the wellstransfected with the unpurified mRNA reprogramming mix died during thereprogramming process, even with the addition of B18R protein. Theaddition of B18R protein did improve the efficiency of reprogramming BJfibroblasts to iPSCs in wells treated with either the RNase III-treatedmRNA reprogramming mix or in wells treated with the HPLC-purified mRNAreprogramming mix.

iPSC Colony Propagation

iPSC colonies from replicate wells reprogrammed with the HPLC-purifiedmRNA reprogramming mix and the RNase III-treated mRNA reprogramming mixwere picked and enzymatically passaged with collagenase onto irradiatedmouse embryonic fibroblast feeder cells in iPS cell maintenance mediumcontaining 10 ng/ml of FGFb. The iPSC colonies were propagated andmaintained a morphology and growth rate as expected for iPSCs for morethan 9 passages in culture, after which they were frozen and stored in afreezer.

Alkaline Phosphatase Staining of iPSC Colonies.

On day 20, plates containing iPSC colonies were fixed with 4%paraformaldehyde and stained to detect alkaline phosphatase-positivecolonies, as previously described. Images of plates of the BJfibroblasts with colonies of cells that stain positively for alkalinephosphatase, a marker for iPSC colonies, are shown in FIG. 28. Thenumbers of alkaline phosphatase-stained iPSC colonies obtained usingψ-modified single-stranded mRNA reprogramming factors from which dsRNAwas removed by either HPLC purification or using the RNase III treatmentdescribed herein so that said mRNA reprogramming factors werepractically free, extremely free or absolutely free of dsRNA, comparedto using unpurified a-modified single-stranded mRNA reprogrammingfactors, are summarized in the Table below.

Immunostaining

After 1 week of storage in the freezer, frozen HPLC-purifiedpseudouridine-modified mRNA-derived iPSCs were thawed, transferred toplates coated with MATRIGEL™ artificial matrix, and propagated in mTeSR™medium. The iPSC colonies were passaged an additional 4 times before aplate was fixed and the cells were immunostained with antibodies toOCT4, TRA1-60, SOX2, TRA1-80 and NANOG pluripotency markerscharacteristic of iPS cells using immunostaining methods as describedpreviously. As shown in FIG. 33, the iPSCs induced from BJ fibroblastsusing HPLC-purified pseudouridine-modified mRNA reprogramming factorswere immunostained positively for these pluripotency markers.

Summary of iPSC Reprogramming with ψ-Modified mRNA Reprogramming Factors

Number of Alkaline Plate B18R Phosphatase- Label in Treatment ProteinDegree of Toxicity Positive iPSC Image Type Used Observed Colonies BelowHPLC- NO Feeder cells dead, but 15 A purified iPSC colonies presentHPLC- YES Feeder cells OK and ~100 B purified iPSC colonies presentRNase III- NO ~50 C Treated RNase III- YES ~100 D Treated Unpurified NOCells dead 0 — Unpurified YES Cells dead 0 —

Pluripotency of iPSCs

Embryoid bodies spontaneously differentiated from picked iPSC coloniesthat had been induced from BJ fibroblasts by HPLC-purified, WI-modifiedmiRNAs encoding the indicated reprogramming factors and then grown for 4to 11 passages in medium in wells coated with MATRIGEL™ matrix asdescribed in EXAMPLE 14. Differentiated cells that stained positivelyfor markers representing all 3 germ layers were observed. For example,cells were observed that expressed the ectoderm marker, neuronal classIII beta-tubulin (TUJ1), the mesoderm markers, alpha-smooth muscle actin(SMA) and desmin, and the endoderm markers, transcription factor SOX17and alpha fetoprotein (AFP).

Example 16. Evaluation of Additional Variables Related to Use of RNaseIII-Treated Modified mRNA Reprogramming Factors Encoding iPS CellInduction Factors for Reprogramming BJ Fibroblasts to iPS CellsMaterials and Methods for Example 16.

The goals of the experiments described in EXAMPLE 16 were to determine(1) whether RNase III-treated mRNA could produce a significant number ofcolonies without the use of an inhibitor of expression of an innateimmune response pathway, such as B18R protein (2) which mRNA encoding acMYC protein—mRNA encoding the wild-type cMYC or mRNA encoding thecMYC(T58A) mutant protein—is more efficient for reprogramming BJfibroblasts into iPSC colonies and (3) whether 10,000 BJ fibrobroblastcells per well is the optimal number of cells for efficientreprogramming to iPSC colonies.

The materials and methods used were similar to those described forEXAMPLES 4-8 above. The mRNA reprogramming factor mix was composed ofψ-modified mRNAs (GAψC) encoding the 3:1:1:1:1 molar mix of OCT4, SOX2,KLF4, LIN28, and cMYC or cMYC(T58A) that were treated with RNase III ina reaction mix containing 1 mM magnesium acetate in order to make themRNA reprogramming factor mix have a low enough level of dsRNA so as tonot interfere with transfection and cell survival. This RNaseIII-treated mRNA reprogramming factor mix was then transfected every dayfor 18 days at a dose of 1.2 micrograms per well per day (unless adifferent mRNA dose is otherwise stated in the respective results table)using the TransIT™ mRNA transfection reagent (Mirus Bio) into 10⁴(unless a different number of cells is stated in the respective resultstable) BJ fibroblasts/well for 8 days in a row in 6-well platescontaining on top of NuFF feeder cells. The experimental variables arelisted in the results tables for each experiment. iPSC colony countswere made by immunostaining live cells with StainAlive™ DyLight™ 488anti-human TRA-1-60 antibody (Stemgent), as described herein, andmanually counting stained iPSC colonies in each visual field using agrid. There was variation in colony size and staining intensity, andsometimes there were “too many colonies to count” (e.g., see FIG. 28),making it challenging or impossible to properly count them. For example,there were more than 300 colonies in each well designated as “too manycolonies to count” or “TMTC”. Therefore, the iPSC colony counts areapproximate.

Results for Example 16.

Efficient Reprogramming of BJ Fibroblasts to iPSC Colonies by RNaseII-Treated Pseudouridine-Modified mRNA Reprogramming Factors in thePresence or Absence of B18R Protein.

iPSC colonies were first detected on Day 13 in two different wells of BJfibroblasts that were transfected with RNAse III-treated (1 mM MgOAc)₂pseudouridine-modified mRNA reprogramming factors in the absence of B18protein in the medium. However, iPSC colonies were also inducedbeginning a day or two later in wells of BJ fibroblasts that weretransfected with RNAse III-treated pseudouridine-modified mRNAreprogramming factors in the presence of 200 ng/ml of B18R recombinanthuman protein in the medium. iPSC colonies induced in the presence ofB18R protein are shown in the image in FIG. 30. In fact, in thisexperiment, it was impossible to determine an effect of B18R proteinbecause the entire well was full of colonies—with hundreds of coloniesper well of a 6-well plate—in both cases and the number of colonies weretoo many to count (TMTC). (If only 100 iPSC colonies had been presentper well, the iPSC induction efficiency would have been 1%)

Type of MYC protein mRNA encoded by mRNA in B18R Type the mRNA ProteiniPSC Colony Used reprogramming mix Used Count on Day 18 GAψC cMYC(T58A)NO TMTC GAψC cMYC(T58A) YES TMTC

Similar experiments in which ω-modified mRNA encoding reprogrammingfactors was used for transfection ±B18R protein in the medium andwherein iPSC colonies generated could be counted are shown below. E.g.,in one experiment evaluating use of mRNA encoding cMYC wild-type proteinversus mRNA encoding cMYC(T58A) mutant protein for reprogramming, moreiPSC colonies were observed on Day 18 when B18R protein was added to themedium one hour prior to every transfection, as shown above. Thisexperiment also indicated that mRNA encoding cMYC(T58A) increased theiPSC colony induction compared to using mRNA encoding wild-type cMYC.

Type of MYC protein mRNA encoded by mRNA in B18R Type the mRNA ProteiniPSC Colony Used reprogramming mix Used Count on Day 18 GAψC cMYCwild-type NO 105 GAψC cMYC wild-type YES 157 GAψC cMYC(T58A) NO 182 GAψCcMYC(T58A) YES TMTC

Another experiment was done to determine whether the number of BJfibroblast cells that were transfected with the RNase III-treatedpseudouridine-modified mRNA reprogramming factors was optimal forreprogramming using either mRNA encoding cMYC wild-type protein or mRNAencoding cMYC(T58A) mutant protein in the mRNA reprogramming mix. If toofew BJ fibroblast cells were being plated, there would be fewer iPSCcolonies induced, whereas if too many BJ fibroblast cells were beingplated (and the cells weren't split mid-reprogramming), the iPSCcolonies would become confluent and couldn't easily be picked, whichwould result in fewer usable iPSC colonies. Based on the result of thisexperiment, plating 5000 to 10,000 passage-number-4 BJ fibroblast cellsper well was ideal, as shown in the results table below. However, insubsequent experiments, we found that later-passage-number BJ fibroblastcells grew more slowly, so it appeared to be better to use more cellswith later-passage BJ fibroblast cells. Thus, the ideal number of cellswill vary by the growth rate of the BJ fibroblasts, with younger cellsusually growing more rapidly and older cells growing more slowly. Basedon the results of this experiment, mRNA encoding the cMYC(T58A) gavetwice as many iPSC colonies under otherwise similar conditions comparedto mRNA encoding the wild-type cMYC protein. Thus, mRNA encoding thecMYC(T58A) mutant protein appeared to be beneficial for iPSC inductionefficiency, as shown in the results table below.

Transfection of too many BJ fibroblasts per well results in fewer iPSCcolonies and mRNA encoding cMYC(T58A) results in more iPSC colonies thanmRNA encoding wild-type cMYC Type of MYC protein mRNA encoded by mRNANumber of BJ Alk Phos-Positive Type in the mRNA Fibroblast Cells iPSCColony Used reprogramming mix Plated Per Well Count on Day 18 GAψC cMYCwild-type  5 × 10³ 80 GAψC cMYC wild-type 10⁴ 105 GAψC cMYC wild-type2.5 × 10⁴ 14 GAψC cMYC(T58A)  5 × 10³ 203 GAψC cMYC(T58A) 10⁴ 182 GAψCcMYC(T58A) 2.5 × 10⁴ 41

Example 17. Reprogramming BJ Fibroblasts to WPS Cells Using RNaseIII-Treated Cap1 Poly-A-Tailed Unmodified (GAUC) mRNAs Encoding OCT4,SOX2, KLF4, LIN28, NANOG and cMYC Reprogramming Factors Materials andMethods for Example 17.

As demonstrated in the above Examples, we have been able to repeatablyand efficiently reprogram BJ fibroblast cells to iPSC colonies using assRNA reprogramming factor mix comprising a 3:1:1:1:1 molar ratio ofpseudouridine-modified and/or 5-methylcytidine-modified mRNAs encodingOCT4, SOX2, KLF4, LIN28, and cMYC, cMYC(T58A) or L-MYC, wherein themodified mRNAs were either HPLC purified or were RNase III treated in areaction mixture containing low levels of divalent magnesium cationsprior to their use in reprogramming. In view of the surprisingly andunexpectedly successful results in reprogramming human or animal somaticcells to iPSC colonies using modified mRNA reprogramming factors thatwere treated with RNase III in the presence of low levels of divalentmagnesium, we decided to evaluate whether it might be possible toreprogram such somatic cells to iPSC colonies using ssRNA reprogrammingfactors comprising unmodified mRNAs encoding OCT4, SOX2, KLF4, LIN28,and cMYC(T58A). The present researchers believe that successfulreprogramming of human or animal somatic cells to iPSC colonies thatcould be propagated in culture for long periods, sufficient to form iPSCcolony lines, using only unmodified ssRNA has not previously beenreported or demonstrated. Thus, in view of the success of the presentresearchers in developing a method for treating in vitro-synthesizedmodified ssRNA with a dsRNA-specific RNase (e.g., RNase III) in order togenerate ssRNAs encoding reprogramming factors with reduced dsRNA,wherein said ssRNAs were intact and functional in reprogramming human oranimal somatic cells to iPSCs, as reported herein, we decided toevaluate whether the same RNase III treatment method described hereincould be used to make unmodified ssRNAs encoding the same reprogrammingfactors that had very low levels of dsRNA, and if so, whether suchtreated ssRNAs could be used to reprogram human or animal somatic cellsto iPS cells. Surprisingly and unexpectedly, this experiment wassuccessful, as reported below.

Thus, a ssRNA reprogramming factor mix comprising a 3:1:1:1:1 molarratio of unmodified cap1 5′capped and poly(A)-tailed (to ˜150-basepoly-A tail length) mRNAs encoding OCT4, SOX2, KLF4, LIN28, andcMYC(T58A) were synthesized by in vitro transcription as describedherein above, except that the RNA was synthesized using only GTP, ATP,CTP, and UTP without use of pseudouridine-5′-triphosphate,5-methylcytidine-5′-triphosphate or another modifiednucleoside-5′-triphosphate and treated with RNase III in a reactioncomprising 1 mM of magnesium acetate, also as described herein above.Dot blot assays with the J2 dsRNA-specific antibody were performed toverify digestion of the dsRNA in the RNase III-treated ssRNAs. Then,10,000 cells per well of BJ fibroblasts on NuFF feeder cells in wells ofa 6-well plate were transfected daily with a dose of either 1.0, 1.2, or1.4 micrograms of the ssRNA reprogramming mix every day for at least 18days using the TransIT™ mRNA transfection reagent (Mirus Bio), all asdescribed in the General Materials and Methods. The experimentalvariables are listed in the results table below for each experiment. OnDay 18 of the reprogramming protocol, the iPSC colony counts were madeby immunostaining live cells with StainAlive™ DyLight™ 488 anti-humanTRA-1-60 antibody (Stemgent), as described in EXAMPLE 16 and elsewhereherein, and manually counting stained iPSC colonies in each visual fieldusing a grid. The results are presented in the table below.

Results for Example 17.

Hundreds of iPSC colonies were generated from unmodified mRNA that wastreated with RNase III using methods as described herein. Type of MYCTotal Alk Phos- Protein Micrograms Positive Encoded by mRNA of mRNAs iniPSC mRNA in the mRNA B18R Reprogramming Colony Type ReprogrammingProtein Mix Per Count Used Mix Used Transfection on Day 18 GAUCcMYC(T58A) NO 1.0 micrograms 262 per well GAUC cMYC(T58A) NO 1.2micrograms 244 per well GAUC cMYC(T58A) NO 1.4 micrograms 88 per wellGAUC cMYC(T58A) YES 1.2 micrograms TMTC per well

As shown in the table above, all three different daily doses of a ssRNAreprogramming mix comprising unmodified mRNAs encoding OCT4, SOX2, KLF4,LIN28, and cMYC(T58A) that were used to transfect BJ fibroblasts for 18days resulted in generation of iPSC colonies. However, this ssRNAreprogramming mix comprising unmodified mRNAs was clearly more toxic tothe cells than the ssRNA reprogramming mix comprisingpseudouridine-modified mRNAs. Thus, one microgram of the reprogrammingmix per well, rather than 1.2 micrograms per well, resulted in lessearly toxicity and, therefore, more cells that survived to theepithelial transition and formed iPSC colonies. When 1.4 micrograms ofreprogramming mix was used daily, most of the feeder cells died,resulting in colonies attached to very few cells as seen in the imagesin FIG. 31.

Colonies induced using RNase III-treated unmodified mRNAs encoding OCT4,SOX2, KLF4, LIN28, and cMYC(T58A) iPSC induction factors were confirmedto be iPSC colonies based on morphology, ability to be propagated forgreater than 16 passages in culture, positive in vivo immunostaining forthe TRA-1-60 using a TRA-1-60 anti-human antibody and StainAlive™DyLight™ 488 (Stemgent), and positive immunofluorescent staining ofparaformaldehyde-fixed cells using antibodies for the iPSC markers OCT4,TRA1-60, NANOG, TRA 1-81 and SSEA4, performed as described in EXAMPLE 13(FIG. 32).

The present researchers believe successful reprogramming of human oranimal somatic cells to iPSC cells using only unmodified ssRNA has notpreviously been reported or demonstrated. Without being bound by theory,we believe that others have not been successful in reprogramming humanor animal cells with unmodified ssRNAs because they have not recognizedthe significance of the low levels of dsRNA contaminants generatedduring in vitro-transcription of ssRNA. Therefore, they did notrecognize the importance of purifying or treating such invitro-synthesized ssRNA in order to remove all or almost all of thedsRNA contaminants. Still further, they have not understood or developeda method for sufficiently purifying or treating said ssRNAs in order toeffectively remove all or almost all of dsRNA contaminants. The presentresearchers have discovered simple, rapid and efficient methods fortreating ssRNAs with a double-strand-specific RNase that results inssRNAs that are free or almost free of dsRNA contaminants. One exampleof such a double-strand-specific RNase that can be used for this purposeis the endoribonuclease, RNase III. However, the present researchersalso discovered, surprisingly and unexpectedly, that treating ssRNA withRNase III using the optimal conditions known in the art since 1968(Robertson, H D et al. 1968) did not sufficiently remove dsRNA so thatthe treated ssRNAs could be used for translation in living cells or forreprogramming living human or animal cells from one state ofdifferentiation to another state of differentiation (e.g., forreprogramming human or animal somatic cells to iPS cells). In fact, whenthe present researchers treated ssRNAs encoding iPSC reprogrammingfactors with RNase III using the method in the literature, all of thecells that were repeatedly transfected with the treated ssRNAs in orderto try to generate iPSCs ultimately died. Detailed analysis of the RNaseIII activity and specificity under different conditions, as described inEXAMPLES 1-9, revealed that the reaction conditions in the literaturedid not sufficiently remove small amounts of dsRNA contaminants from invitro-transcribed ssRNA for use in introducing into living cells andthat those conditions also resulted in significant degradation of thetreated ssRNAs that the present researchers desired to be translated inthe living cells. In other words, not only did the RNase III method inthe literature fail to sufficiently remove the undesired dsRNA, it alsodestroyed a portion of the desired ssRNAs that encoded the proteins ofinterest. Next, the present researchers tried to modify the conditionsthat were suggested by various authors who had developed or used theRNase III method in the literature, including for example, changing thetype or concentration of monovalent salt, the pH, and the amount ofenzyme used, but to no avail. Thus, although the literature pertainingto RNase III suggested that changing the concentration of the monovalentsalt in the RNase III reaction might be beneficial, the presentinventors tried ranges of concentrations of different monovalent saltswithout success. Changes of variables suggested in the literature didnot result in sufficient removal of the dsRNA for the ssRNAs to be usedfor reprogramming living cells, did not sufficiently reduce the toxicityof the ssRNAs, and still result in damage or destruction of at least aportion of the desired ssRNAs.

Without being bound by theory, the present researchers believe that thehigh cellular toxicity is due to the extremely low levels of dsRNA thatare detected by the innate immune response and other RNA sensors thatare present in human and animal cells to protect those cells frominfection by dsRNA viruses and other pathogens. Thus, due to the extremesensitivity of human or animal cells to dsRNA that is introduced intothose cells, a method that is suitable for reducing dsRNA from ssRNAsfor in vitro applications is not necessarily sufficient for makingssRNAs for introducing into living human or animal cells. The innateimmune response and other RNA sensors (e.g., toll like receptors, e.g.,TLR3, interferons, and other such sensors) are induced to higher levelsif even a certain small quantity of dsRNA is introduced into said cells.Still further, inductions of certain RNA sensors may sensitize the cellsto future introductions of the same ssRNA. In addition, the toxiceffects of the innate immune response may be cumulative. For example,repeated introductions of dsRNA induces interferons, which results inphosphorylation of PKR, which results in inhibition of protein synthesisin the cells, which, in turn, can lead to prolonged toxicity to thecells and, ultimately, programmed cell death (apoptosis). Thus, withrespect to the methods for reprogramming human somatic cells to iPScells, wherein one introduces ssRNAs encoding reprogramming factorsevery day for 18 or more days in order to generate the iPS cells, theinnate immune response and other RNA sensor responses are induced eachtime the ssRNAs encoding reprogramming factors are introduced into thecells.

Example 18. Feeder-Free Reprogramming of 1079 Fibroblast Cells to iPSCells Using Single-Stranded mRNA Encoding iPSC Induction FactorsMaterials and Methods for Example 18

In this EXAMPLE 18, 1079 fibroblast cells (ATCC, Manassas, Va.) wereplated at cell densities of 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, or 5×10⁴ cellsper well in 6-well tissue culture plates coated with 83 ng-per-well ofMATRIGEL™ GFR matrix (BD Biosciences, San Jose, Calif.) in fibroblastmedium composed of Advanced MEM (Invitrogen, Carlsbad, Calif.)supplemented with 10% FBS (Fisher) and 2 mM GLUTAMAX-I (InvitrogenCarlsbad, Calif.) prior to their use for reprogramming.

On the following day, the medium was changed to reprogramming medium,composed of DMEM/F12 (Invitrogen Carlsbad, Calif.) supplemented with 20%KNOCKOUT™ Serum Replacement (Invitrogen Carlsbad, Calif.), 2 mMGLUTAMAX-I (Invitrogen), 0.1 mM non-essential amino acids solution(Invitrogen Carlsbad, Calif.), 100 ng/ml basic human recombinant FGF(Invitrogen Carlsbad, Calif.), 2 micromolar TGFβ inhibitor STEMOLECULESB431542 (Stemgent, Cambridge, Mass.), 0.5 micromolar MEK inhibitorSTEMOLECULE PD0325901 (Stemgent Cambridge, Mass.), and 10 ng/mlrecombinant mouse LIF (Invitrogen Carlsbad, Calif.) withpenicillin-streptomycin antibiotics. Reprogramming was performed withpseudouridine-containing, RNase III-treated mRNAs encoding OCT4, SOX2,KLF4, LIN28, and cMYC(T58A) iPSC induction factors in the feeder-freereprogramming medium as previously described for BJ fibroblasts inEXAMPLE 11.

Results for Example 18.

As previously found using BJ fibroblasts in EXAMPLE 11, 1079 fibroblastcells were also successfully reprogrammed into iPS cells using a3:1:1:1:1 mixture of pseudouridine-containing, RNase III-treated mRNAsencoding the reprogramming factors, OCT4, SOX2, KLF4, LIN28, andcMYC(T58A) in the feeder-free reprogramming medium as described above.

Reprogramming of the 1079 fibroblast cells was observed in wells platedwith 1×10⁴ and 2×10⁴ cells per well. More iPSC colonies were induced atthe lowest cell density tested (1×10⁴ cells per well), with 24 iPSCcolonies observed, versus only 8 iPSC colonies in the well plated with2×10⁴ cells. At higher cell densities, no reprogramming was observedwith the rapidly-growing 1079 fibroblast cells due to the cellsovergrowing the wells before reprogramming occurred.

Example 19. Variation of Stoichiometry of mRNAs Encoding iPSCReprogramming Factors

In this experiment, we compared reprogramming of human fibroblasts toiPSCs using an mRNA mix encoding KLF4, LIN28, cMYC(T58A), OCT4 and SOX2in in a molar ratio of 3:1:1:3:1 with the previously describe mRNA mixhaving a molar ratio of 1:1:1:3:1.

Materials and Methods for Example 19.

10⁴ BJ fibroblasts (passage 6) were plated on 4×10⁵ NuFF feeder cells inPluriton reprogramming media as previously described. The mediacontaining RNase Inhibitor was changed prior to daily transfections.mRNA mixes comprising RNase III-treated (in 2 mM Mg²⁺) cap1,poly(A)-tailed (˜150 As), pseudouridine-modified mRNA encoding KLF4,LIN28, cMYC(T58A), OCT4 and SOX2 were synthesized as previouslydescribed. In Experiment 19-1, the mRNA mixes were diluted in 60microliters of Stemfect Buffer, combined with the Stemfect TransfectionReagent diluted in 60 microliters of Stemfect Buffer, and the mix wasincubated at room temperature for 15 minutes and added dropwise to thecells during each of eighteen daily transfections. In Experiment 19-2,either 1.0 microgram, 1.2 microgram or 1.4 microgram of each mRNA mixwas transfected with 4, 4.8 or 5.6 microliters of Stemfect TransfectionReagent, respectively, and only 15 transfections were performed. ThemRNA reprogramming mixes were produced with mRNA encoding KLF4 at 1×, 2×and 3× molar ratios compared to the LIN28, cMYC(T58A), and SOX2 mRNAs(i.e., with 1:1:1:3:1; 2:1:1:3:1; and 3:1:1:3:1 stoichiometries).

After completion of all transfections, wells with iPSC colonies werefixed and stained with alkaline phosphatase after representativecolonies were picked for expansion. Alkaline phosphatase-positive colonycounts obtained and imaged.

Experiment 19-1: No. of Alkaline Phosphatase-Positive Colonies Obtained.

Alk AMT Transfn Phos- mRNA of Reagent Positive Reprogramming RNA and volColony Well Mix mRNA Type (μg) (μl) Count No. 4F KMO₃S Ψ RIII Cap 1 1.0SF 4 3 1 4F KMO₃S Ψ RIII Cap 1 1.2 SF 4.8 29 2 4F KMO₃S Ψ RIII Cap 1 1.4SF 5.6 44 3 5F KLMO₃S Ψ RIII Cap 1 1.0 SF 4 13 7 5F KLMO₃S Ψ RIII Cap 11.2 SF 4.8 57 8 6F KLMNO₃S Ψ RIII Cap 1 1.0 SF 4 39 9 (+Nanog) 6FKLMNO₃S Ψ RIII Cap 1 1.2 SF 4.8 109 10 (+Nanog) 5F K₃LMO₃S Ψ RIII Cap 11.0 SF 4 40 11 5F K₃LMO₃S Ψ RIII Cap 1 1.2 SF 4.8 148 12 TransfectionOptim. Stemfect 5F KLMO₃S Ψ RIII Cap 1 1.0 SF 3 6 13 5F KLMO₃S Ψ RIIICap 1 1.0 SF 4 16 14 5F KLMO₃S Ψ RIII Cap 1 1.0 SF 5 31 15 5F KLMO₃S ΨRIII Cap 1 1.0 SF 6 7 16

Summary of Results for Example 19 Experiment 19-1.

As seen previously, including miRNA encoding NANOG in the mRNA mixresulted in more iPSC colonies than the 5 factor mix that did notinclude NANOG. The 6 factor mix (KLMNO₃S) produced the most colonies andthe earliest colonies.

An interesting result from this experiment was the effect of increasingthe amount of miRNA encoding KLF4 in the reprogramming mRNA mix. The 5factor mix using a 3:1:1:311 ratio of KLMO and S resulted in the firstcolonies, the largest colonies, and the most colonies. The 6 factor mixwas the best.

The most beneficial effect of using more mRNA encoding KLF4 was theuniform morphology of the iPSC colonies generated. With other mRNA mixeswith KLF4 mRNA representing a 1-fold molar ratio, we have observed iPSCcolonies of varying size and cell stage; the first iPSC colonies haveoften begun to differentiate before the last transfection was performed.Some of the iPSC colonies also have exhibited what the presentresearchers believe to be incompletely reprogrammed cells surroundingthem. Some representative images of typical iPSC colonies obtained areshown in FIG. 38 and FIG. 39.

As can be seen in FIG. 38, some of the 5-factor pseudouridine-modified,RNase III-treated KLMO3S (1:1:1:3:1) iPSCs are regular in shape, butmany of the cells are not. There are larger epithelial cells aroundthese two colonies. The scattered cells around the periphery are thefeeder cells.

As can be seen in FIG. 39, all of the 5 factor pseudouridine-modified,RNase III-treated K₃LMO₃S (3:1:1:3:1) iPSCs had more regular borders.These cells also tended to kill off the feeder cells surrounding theiPSC colonies. These colonies were larger and easier to pick forpropagation because they were more uniform.

Having control over the factor stoichiometry is one of the benefits ofusing mRNA for reprogramming, such as to find the ideal ratios of mRNAsencoding different reprogramming factors to achieve a particular effect.

Currently the only drawback to elevating the KLF4 level is the feedercell layer death, but because the colonies thrived and were easy to workwith, this may actually be a benefit. As is shown in FIG. 40, whenalkaline phosphatase stained, the benefits to the colonies resultingfrom the elevated KLF4 mRNA in the mRNA mix can clearly be seen (seeFIG. 40 B).

Experiment 19-2: Effect of Amount of mRNA Encoding KLF4 in 5-Factor mRNAMixes Comprising RNase III-Treated CAP1 GAψC-mRNAs on Induction of iPSCsfrom BJ Fibroblasts

Total Amount of Epith No. of Alk. Amount mRNA Klf4 mRNA CellsPhos.-Positive Well of mRNA Mix in Mix Noted Observations Colonies # 1.0μg KLMO₃S 1X 149  28 1.0 μg K₂LMO₃S 2X Very Early 181  19 1.0 μg K₃LMO₃S3X Very Early 73 22 1.2 μg KLMO₃S 1X TMTCA 400+ 29 1.2 μg K₂LMO₃S 2XEarliest TMTCA 400+ 20 1.2 μg K₃LMO₃S 3X Earliest 194  23 1.4 μg KLMO₃S1X TMTCA 400+ 30 1.4 μg K₂LMO₃S 2X Earliest TMTCA 400+ 21 1.4 μg K₃LMO₃S3X Earliest ~300  24 *TMTCA = Too many Alk Phos-Positive colonies tocount accurately; N/A = Not Applicable.

Summary of Results for Example 19 Experiment 19-2.

There was definitely a reproducible benefit to increasing the amount ofmRNA encoding KLF4 in the mRNA reprogramming mixes. The K₃LMO₃S mRNA mixcaused feeder cell death as seen in the previous experiments. However,the K₂LMO₃S and K₃LMO₃S mixes resulted in earlier epithelial cellformation and iPSC colonies that were reproducibly larger and easier topick. In this experiment, the K₂LMO₃S mRNA mix resulted early inductionof iPSC colonies with less cell death (including feeder cell death) andhigher numbers of iPSC colonies than the K₃LMO₃S mRNA mix, so that fewertransfections could be performed to obtain good numbers of iPSC coloniesefficiently.

Example 20. Effect of Different Caps and Other Variations on iPSCReprogramming Materials and Methods for Example 20

Sets of different reprogramming mRNAs were synthesized that varied bycap, nucleotide composition and RNAse III (RIII) treatment. APEX™phosphatase (Epicentre, Madison, Wis., USA) was used to treat someco-transcriptionally ARCA-capped mRNAs (see table below). ReprogrammingmRNAs encoding KLF4 (K), LIN28 (L), cMYC(T58A) (M), OCT4 (0) and SOX2(S)were mixed to maintain a 3-fold molar excess of OCT4 over the otherfactors, regardless of the number of factors encoded in the mRNA mixes.

10⁴ BJ fibroblasts (passage 6) were plated on 4×10⁵ NuFF feeder cells inPluriton reprogramming media as previously described. When B18R proteinwas used, the medium was changed four hours prior to the firsttransfection and B18R protein (200 ng/ml media) was added to the well.For subsequent transfections, the B18R protein and RNase Inhibitor(0.5U/ml medium) was added to cells in fresh medium, which was changedprior to daily transfections. Eighteen transfections were performedusing 1.2 micrograms of mRNA diluted in 60 microliters of StemfectBuffer combined with 4.8 microliters of the Stemfect transfectionreagent diluted in 60 microliters of Stemfect buffer. Each mRNA mix wasincubated at room temperature for 15 minutes and added dropwise to thecells.

The colonies were fixed and stained with alkaline phosphatase afterrepresentative colonies were picked for expansion. Colony counts wereperformed on fixed, alkaline phosphatase-positive cells, as presented inthe table below.

Results for Example 20 No. of Alkaline Phosphatase-Positive ColoniesObtained:

Alk Phos- AMT Positive of B18R iPSC Cap RNA Protein Colony Well Type(μg) Used Count No. mRNA Reprogramming Mix unmod. RIII 4F KMOS Cap 1 1.2NO 18 4 unmod. RIII 5F KLMOS Cap 1 1.2 NO 111 5 unmod. RIII 6F KLMNOSCap 1 1.2 NO 140 6 ψ-modified mRNA Reprogramming Mix ψ RIII 5F KLMOS Cap0 1.2 NO 223 9 ψ RIII 5F KLMOS Cap 0 1.2 YES 162 10 ψ RIII 5F KLMOS Cap1 1.2 NO 214 13 ψ RIII 5F KLMOS Cap 1 1.2 YES 115 14 ψ RIII 5F KLMOSARCA 1.2 NO 317 17 ψ RIII 5F KLMOS ARCA 1.2 YES 292 18 ψ & m⁵C 5F KLMOSARCA + 1.2 NO 5 21 (no RIII) Phos ψ & m⁵C 5F KLMOS ARCA + 1.2 YES 102 22(no RIII) Phos (RIII = RNase III-treated. O is in 3-fold molar excessover other reprogramming mRNAs.)No. of Alkaline Phosphatase-Positive Colonies Obtained: iPSC ColonyCounts for Each Well are Given Below:

No. of AMT Alk of B18R Phos- Cap RNA Protein Positive Well mRNA mix Type(μg) Used Colonies No. unmod. RIII 4F KMOS Cap 1 1.0 NO 3 1 unmod. RIII5F KLMOS Cap 1 1.0 NO 18 2 unmod. RIII 6F Cap 1 1.0 NO 34 3 KLMNOSunmod. RIII 4F KMOS Cap 1 1.2 NO 18 4 unmod. RIII 5F KLMOS Cap 1 1.2 NO111 5 unmod. RIII 6F Cap 1 1.2 NO 140 6 KLMNOS ψ RIII 5F KLMOS Cap 0 1.0NO 66 7 ψ RIII 5F KLMOS Cap 0 1.0 YES 38 8 ψ RIII 5F KLMOS Cap 0 1.2 NO223 9 ψ RIII 5F KLMOS Cap 0 1.2 YES 162 10 ψ RIII 5F KLMOS Cap 1 1.0 NO73 11 ψ RIII 5F KLMOS Cap 1 1.0 YES 70 12 ψ RIII 5F KLMOS Cap 1 1.2 NO214 13 ψ RIII 5F KLMOS Cap 1 1.2 YES 115 14 NO ψ RIII 5F KLMOS ARCA 1..0NO 120 15 ψ RIII 5F KLMOS ARCA 1.0 YES 45 16 ψ RIII 5F KLMOS ARCA 1.2 NO317 17 ψ RIII 5F KLMOS ARCA 1.2 YES 292 18 ψ m⁵C 5F KLMOS ARCA + 1.0 NO0 19 (no RIII) Phos ψ m⁵C 5F KLMOS ARCA + 1.0 YES 34 20 (no RIII) Phos ψm⁵C 5F KLMOS ARCA + 1.2 NO 5 21 (no RIII) Phos ψ m⁵C 5F KLMOS ARCA + 1.2YES 102 22 (no RIII) Phos (RIII = RNase III-treated. O is always in3-fold molar excess over other reprogramming mRNAs.)

Summary of Selected Results for Example 20.

Induced pluripotent stem cell colonies were generated using unmodifiedmRNAs encoding only 4 factors (KMOS). Some colonies were picked toexpand and maintain. The cells tolerated the Stemfect RNA Transfectionreagent with unmodified mRNA without a media change post-transfection.B18R protein consistently decreased the colony counts from RNaseIII-treated-, ψ-modified mRNA mixes. The only benefit for use of B18Rprotein was with dual (ψ- and m5C-) modified mRNA that wasn't treatedwith RNAse III. As can be seen in FIG. 43 C, fewer colonies wereobtained with the dual modified (ψ- and m5C-)ARCA-co-transcriptionally-capped mRNA mix, even though it wasphosphatased and B18R protein was added to the medium.

The number of colonies from Cap0 and Cap1 RIII-treated, ψ-modified mRNAmixes were very similar (see FIG. 42 and FIG. 43 A).

As is shown in FIG. 43 B. the ARCA-capped, ψ-modified RIII-treated mRNAmix produced iPSC colonies from post-transcriptionally-capped mRNA mixesin this experiment. The tail lengths of these mRNA were slightly shorterthan the Cap0 and Cap1 mRNA tails. Denaturing gel tail lengthcomparisons aren't as accurate on pseudoU-modified mRNA, but the tailsappeared to be ˜120 bases on the Cap0 and Cap1 mRNAs and at least 100bases on the ARCA-capped mRNAs. Some colonies were picked and removedfor propagation before staining.

Example 21. Additional Studies on Effect of Different Caps and OtherVariations on iPSC Reprogramming Materials and Methods for Example 21

mRNAs encoding 5 reprogramming factors (KLM_(T58A)OS) were synthesizedusing standard unmodified GAUC NTPs. The RNAs were either synthesizedco-transcriptionally capped with the ARCA cap analog, orpost-transcriptionally capped to either have a Cap0 or Cap1. All of themRNAs were poly(A) tailed using poly(A) polymerase to a length of ˜150As. Some of the ARCA-capped mRNAs were also treated with Apexphosphatase.

5-Factor reprogramming mixes were made (KLM_(T58A)OS with standard1:1:1:3:1 stoichiometry) and 1.2 micrograms of each mRNA mix wastransfected with 4.8 microliters Stemgent's STEMFECT™ TransfectionReagent daily into 10⁴ BJ fibroblasts passage 5 plated on 4×10⁵ NuFFcells. Some wells had 200 ng or 400 ng B18R protein added per ml ofmedium. After 18 transfections, the cells were grown for 2 more days, afew iPSC colonies were picked and the rest were stained for alkalinephosphatase activity to count iPSC colonies.

Experiment 1: No. of Alkaline Phosphatase-Positive Colonies from EachType of mRNA Mix.

Treated No. of RIII with B18R Alk Phos- Cap RNase Buffer APEX ™ ProteinPositive Well Type III Mg Concn Phosphatase Used Observations ColoniesNo. ARCA (co-trans) NO N/A NO NO Cells died  0 1 ARCA NO N/A YES NOCells died  0 2 ARCA NO N/A YES YES 200 ng 106 3 ARCA YES 2 mM NO NO  04 ARCA YES 2 mM YES NO  0 5 ARCA YES 2 mM YES YES 200 ng 278 6 ARCA YES2 mM NO YES 200 ng  90 7 ARCA YES 2 mM NO YES 400 ng  93 8 Cap0 NO N/AN/A NO Cells died  0 9 Cap0 YES 1 mM N/A NO  0 10 Cap0 YES 2 mM N/A NO 0 11 Cap0 YES 1 mM N/A YES 200 ng  400+ 12 Cap0 YES 2 mM N/A YES 200 ng283 13 Cap0 YES 1 mM N/A YES 400 ng 252 14 Cap0 YES 2 mM N/A YES 400 ng344 15 Cap1 NO N/A N/A NO Cells died  0 16 Cap1 YES 1 mM N/A NO 394 17Cap1 YES 2 mM N/A NO 386 18 Cap1 YES 1 mM N/A YES 200 ng  400+ 19 Cap1YES 2 mM N/A YES 200 ng  400+ 20 Cap1 YES 1 mM N/A YES 400 ng  400+ 21Cap1 YES 2 mM N/A YES 400 ng  400+ 22

Results for Example 21.

ARCA Results

Unmodified, ARCA co-transcriptionally capped mRNA produced iPSC coloniesif the mRNA was treated with phosphatase and the cells are treated withB18R (well #3). The Stemfect Transfection Reagent's low toxicity mayalso play a role in making this possible. RNase III-treatment of theARCA mRNA was not enough to produce colonies (wells #4 & 5) unless thecells were also treated with B18R (wells 6, 7 & 8). RNase III-treatmentsignificantly increased the number of iPSC colonies obtained withARCA+phosphatase treatment+B18R in the medium (well #6 versus 3). TheAPex Phosphatase treatment seemed to significantly improve the iPSCcolony count (well 6, versus Wells 7 & 8). Increasing the B18Rconcentration from 200 ng/ml of media to 400 ng/ml of media did notincrease the iPSC colony count (well 8 versus well 7).

Cap0 Results

In this experiment, RNase III-treatment of the Cap0 mRNA was not enoughto produce iPSC colonies (wells #10 & 11) unless the cells were alsotreated with B18R (wells 12 to 15). 2× B18R did not seem to increase thenumber of iPSC colonies, but the results were mixed. In one case, 1 mMMg(OAc)₂ was better than 2 mM in the RNase III buffer; in one case itwas not (wells 12 to 15).

Cap1 Results

Use of Cap1 unmodified mRNAs that were treated with RNase III wassufficient to produce iPSC colonies, whereas RNase III-treated Cap0- orARCA-capped unmodified mRNAs did not induce iPSC colonies. B18R proteindid seem to increase the number of iPSC colonies from RNase III-treated,Cap1 unmodified mRNAs. No difference could be determined when 1 or 2 mMMg2+ was used in the RNase III buffer; the iPSC colony counts wereeither similar or there were too many colonies to count.

Comparisons

Cap0 and Cap1 unmodified mRNA mixes produced more iPSC colonies than theARCA-capped mRNA mRNA mixes. Cap1, RNAse III-treated unmodified mRNAreprogramming mixes induced iPSC colonies without B18R protein, but Cap0and ARCA mRNA mixes did not. If RNAse III treatment and B18R proteinwere used together with Cap0 or Cap1 unmodified mRNAs, numerous iPSCcolonies were induced. Picked and propagated alkalinephosphatase-positive iPSC colonies from six different wells (wellnumbers 3, 6, 13, 17, 18, and 20) all stained positive forimmunofluorescent TRA1-60, SOX2, OCT4, SSEA4, and NANOG pluripotencymarkers.

Followup Experiment

The same RNase III-treated (with 1 or 2 mM Mg2+) unmodified mRNAreprogramming mixes containing mRNAs encoding 5 reprogramming factors(KLM_(T58A)OS) (wherein the mRNAs were either co-transcriptionallycapped with the ARCA cap analog, or enzymatically cappedpost-transcriptionally to generate either Cap0 or Cap1 mRNA, andenzymatically poly-A tailed (˜150 A nucleotides) using poly(A)polymerase) were used for reprogramming of BJ fibroblasts as describedabove in this EXAMPLE 21, except that 0.5 micrograms of each mRNA mix,complexed with 2.5 microliters of Invitrogen's RNAiMAX™ transfectionreagent instead of Stemgent's STEMFECT reagent, was transfected dailyfor 18 days into 5×10³ BJ fibroblasts (passage 5) plated on 2.5×10⁵ NuFFcells in 12-well plates (rather than 6-well plates). Some wells also had200 or 400 ngs of B18R protein added per ml of medium. As a positivecontrol for reprogramming, an RNase III (with 2 mM Mg2+)-treatedpseudouridine-modified mRNA mix encoding the 5 KLM_(T58A)OSreprogramming factors was also similarly transfected into BJ fibroblastcells in one well. The cells died in all but two of the wells by the endof the transfections, apparently because the RNAiMAX transfectionreagent was too toxic for the cells under the conditions used. Only afew alkaline phosphatase-positive colonies were generated using theRNase III-treated pseudouridine-modified mRNA positive control mix. Noother conclusions could be made from this experiment.

Example 22. Reprograming of Human Neonatal Keratinocytes (HEKn) to iPSCsUsing RNAse III-Treated, Cap1, Poly(A)-Tailed mRNAs Encoding ProteinReprogramming Factors is Reproducible and Very Efficient Materials andMethods for Example 22

Keratinocyte Reprogramming Protocol

In general, many steps of the Keratinocyte Reprogramming Protocoldeveloped and used herein are similar to the steps in the protocol forreprogramming fibroblasts to iPSCs in the section entitled “DetailedDescription of the Reprogramming Method for EXAMPLE 14,” with someadditional steps as described herein below.

Primary neonatal keratinocytes are propagated in a serum-free, lowcalcium medium that promotes a highly proliferative and undifferentiatedstate. In the presence of physiological levels of calcium, the cellsterminally differentiate into fully stratified epidermis. In order toreprogram these cells most efficiently, the first 3 transfections areperformed with the cells growing in low calcium keratinocyte medium(EpiLife Medium with Human Keratinocyte Growth Supplement from LifeTechnologies) without feeder cells. 2×10⁵ HEKn cells are plated andtransfected daily 3 times. Four hours after the third transfection, thecells are treated with 0.025% trypsin/EDTA solution and are transferredto NuFF cell feeder layers in Pluriton reprogramming medium as describedpreviously for fibroblast reprogramming. From this point on the cellsare transfected in PLURITON™ Reprogramming Medium and Conditioned Mediumas described previously.

More specifically, 2×10⁵ HEKn cells (passage 4) were plated (on plastic)per well of a 6-well dish. The cells were maintained in EpiLife mediumwith 60 micromolar calcium and supplemented with Human KeratinocyteGrowth Supplement (HKGS) both from Cascade Biologics (sold through LifeTechnologies). The cells were transfected daily with 4 microliters ofStemfect RNA Transfection Reagent per microgram of mRNA mix. TheStemfect reagent is diluted in 60 microliters of its own buffer. The 1.2micrograms of mRNA is also diluted in 60 microliters of Stemfect Buffer.The mixes are combined, incubated at room temperature for 15 minutes andadded drop-wise to the cells. The medium was changed daily before thetransfection and 0.5 units of SCRIPTGUARD™ RNase inhibitor (CELLSCRIPT,INC., Madison, Wis., USA) were added per ml of medium. Thus, in someembodiments, an RNase inhibitor (e.g., SCRIPTGUARD™ RNase inhibitor isadded with the RNA comprising ssRNA or mRNA encoding one or moreproteins for inducing a biological or biochemical effect (e.g., forreprogramming a somatic cell, e.g., a keratinocyte, to an iPSC).

The first 3 transfections were performed with the cells maintained inEpiLife low calcium medium. Four hours after the third transfection thecells were trypsinized with 0.025% trypsin/EDTA solution and the cellsfrom each well were plated onto NuFF feeder cells in Pluritonreprogramming medium with standard levels of supplement andpenicillin-streptomycin as previously described. The next 6transfections were performed in Pluriton Reprogramming medium and thefinal 9 transfections were performed with cells maintained in NuFFconditioned Pluriton medium.

mRNAs were synthesized either using standard unmodified ATP, CTP, GTPand UTP or using ATP, CTP, GTP and ψTP. All were capped to make a Cap1structure and tailed using poly(A) polymerase as previously described.In one experiment, HEKn human neonatal keratinocytes were reprogrammedusing the reprogramming method of the present invention by transfecting2×10⁵ HEKn cells with 1-1.5 micrograms of a pseudoU-modified, RNaseIII-treated, mRNA mix of KLM_(T58A)OS (1:1:1:3:1) daily for 14 days.Alkaline phosphatase-positive colonies indicative of iPSCs were obtainedand, following picking and passaging, tested colonies were also positivefor the immunofluorescent pluripotency markers TRA1-60, SOX2, OCT4,SSEA4, and NANOG. Further, when some of these iPSC colonies were allowedto differentiate in the embryoid body spontaneous differentiationprotocol, selected differentiated cells expressed markers of all threegerm layers, including endoderm (AFP and SOX17), mesoderm (SMA andDesmin), and ectoderm (class III beta-tubulin) when cells were fixed andprocessed for immunofluorescence with antibodies that recognized thosemarkers. This led us to perform additional experiments on reprogrammingof HEKn cells using different mRNA reprogramming mixes.

A 6-factor mRNA reprogramming mix (KLM_(T58A)NOS) was made with RNaseIII-treated unmodified mRNAs for reprogramming. Reprogramming with mRNAencoding NANOG in the mRNA mix consistently yields more and earlier iPSCcolonies than 5-factor mixes. A second 6-factor mRNA reprogramming mixencoding the same factors was made with RNase III-treated, ψ-modifiedmRNAs. These mRNA mixes were compared in reprogramming to a 5-factormRNA reprogramming mix made with a 3 times higher molar ratio of KLF4mRNA (3:1:1:3:1), which had resulted in more iPSC colonies, more uniformcolonies and earlier colonies than a 1:1:1:3:1 mRNA mix.

1.2 micrograms or 1.5 micrograms of each mRNA mix was transfected with4.8 microliters or 6 microliters of Stemgent's Stemfect TransfectionReagent daily into 2×10⁵ HEKn cells (passage 5) plated on plastic forthe first 2, 3 or 4 days and then trypsinized and plated on 4×10⁵ NuFFcells 4 hours post-transfection. By day 4, the HEKn cells that werestill growing on plastic were already confluent. This makes themterminally differentiate, so the day 4 transfer wells were dropped fromthe experiment.

After only 14 daily transfections, many iPSC colonies were apparent sono more transfections were performed and the cells were grown for 2 moredays, colonies were picked, fixed, stained for alkaline phosphatase andcounted.

The Table Below is a Summary of the Colony Counts Resulting from EachType of mRNA Mix.

Number of Amount Number Alk Phos- of RNA of Days positive RNaseTransfected before iPSC Colonies III Type Daily plated on after 14 WellSubstitutions Treated of mix (μg) NuFFs Transfections No. Unmodified Yes6F 1.2 2 1 1 KLMNO3S Unmodified Yes 6F 1.5 2 1 2 KLMNO3S PseudoU (Ψ) Yes6F 1.2 2 1 3 KLMNO3S PseudoU (Ψ) Yes 6F 1.5 2 5 4 KLMNO3S PseudoU (Ψ)Yes 5F 1.2 2 12  5 K3LMO3S PseudoU (Ψ) Yes 5F 1.5 2 106  6 K3LMO3SUnmodified Yes 6F 1.2 3 4 7 KLMNO3S Unmodified Yes 6F 1.5 3 5 8 KLMNO3SPseudoU (Ψ) Yes 6F 1.2 3 26  9 KLMNO3S PseudoU (Ψ) Yes 6F 1.5 3 111  10KLMNO3S PseudoU (Ψ) Yes 5F 1.2 3 195  11 K3LMO3S PseudoU (Ψ) Yes 5F 1.53 400+  12 K3LMO3S

Results for Example 22

Representative alkaline phosphatase-positive colonies, which wereobserved in all of the wells, were picked and propagated for furtherIPSC characterization. Reprogramming of primary human neonatalkeratinocytes was reproducible and efficient. In this Example, three wasthe best number of transfections to perform before the cells were platedon feeder cells (for this number of starting keratinocyte cells). Weobserved that fewer than 105 HEKn cells should be plated per 6 well toavoid overgrowth and terminal differentiation of the target cells. Iftoo many cells were plated, the cells wouldn't replate well on feedercells. More RNA and Stemfect reagent needed to be used for highefficiency reprogramming. A total of 1.5 micrograms comprising all ofthe mRNAs per well was more efficient than 1.2 micrograms per well.Using more mRNA encoding KLF4 in the mix produced more iPSC colonies infewer days. Induction of iPSC colonies with RNAse III-treated unmodifiedmRNAs were observed, but reprogramming was inefficient. Picked andpropagated alkaline phosphatase-positive colonies from well numbers 2, 9and 11 all stained positive for immunofluorescent TRA1-60, SOX2, OCT4,SSEA4, and NANOG pluripotency markers.

Example 23. Feeder-Free Reprogramming Using Unmodified orPseudouridine-Modified mRNAs Encoding Reprogramming Factors Materialsand Methods for Example 23:

Feeder-free reprogramming was performed as previously described using BJfibroblasts at passage 4, except 16 consecutive transfections were donerather than 18 transfections. Cells were transfected with cap1 mRNAencoding the 5 reprogramming factors, OCT4, SOX2, KLF4, LIN28 and cMYC(T58A). The mRNA used contained one of the following: unmodified NTPswith RNase III treatment after IVT, unmodified NTPs without RNAse IIItreatment after IVT, or a pseudouridine only substitution with RNAse IIItreatment after IVT. RNase III treatment of the mRNAs was performed aspreviously described. Colony counts were done on day 18 based onmorphology.

Results for Example 23.

No iPSC colonies were observed when unmodified mRNAs were used forreprogramming without RNAse III treatment of the mRNAs after IVT.Induced pluripotent stem cell colonies were observed in wells treatedwith unmodified mRNAs that were RNase III-treated after IVT. However,more colonies were seen if pseudouridine was substituted for uridine inthe mRNAs.

Number of iPSC Colonies Treatment Observed by Morphology Unmodified +RNase III 27 Unmodified − RNase III 0 ΨTP modified + RNase III 51

Example 24. Differentiation of Reprogrammed iPSC Colonies Induced fromBJ Fibroblasts Using RNase III-Treated ψ-Modified mRNAs Encoding iPSCInduction Factors in Feeder-Free Medium Materials and Methods forExample 24.

Feeder-free reprogrammed iPS cells using Ψ-substituted mRNA encoding thefive reprogramming factors, OCT4, SOX2, KLF4, LIN28, and cMYC were putthrough the cardiomyocyte differentiation protocol as previouslydescribed. Movies of beating aggregates were recorded. Aggregates,beating and non-beating, were dissociated using 10× trypsin (Invitrogen,Carlsbad, Calif.). Briefly, aggregates were resuspended in 10× trypsin,incubated at 37° C. for 5 minutes, and broken up using a pipet. Thetrypsin was neutralized with cardiomyocyte maintenance medium, and thecells were spun down at 1,200 rpms for 5 minutes. Cells were resuspendedin cardiomyocyte maintenance medium and plated onto 6 well tissueculture plates pre-coated with 0.1% gelatin. Media was changed thefollowing 2 days. Cells were then fixed and stained for class IIIbeta-tubulin, cardiac troponinT, and sox17.

Results for Example 24.

Feeder-free reprogrammed iPSCs were put into cardiomyocytedifferentiation. Beating aggregates were observed on day 12 ofdifferentiation, and videos of the beating aggregates were recorded.After completion of the differentiation protocol, aggregates, bothbeating and non-beating, were dissociated and plated onto gelatin to seeif cells originating from the other 2 germ layers also formed from theseiPSCs. Cells staining positive for a neuronal makers, class IIIbeta-tubulin, were observed indicating the potential of the iPSCs todifferentiate into ectoderm originating cells. As is shown in FIG. 45,cells staining positive for SOX17, a transcription factor found in cellsof the endoderm lineage, were also observed. Cells were also seen thatstained positive for cardiac troponinT, which is a marker ofcardiomyocytes, which have a mesoderm origin. Thus, the feeder-freereprogrammed iPSCs were able to differentiate into cells of all 3 germlayers.

Example 25. Forward Differentiation of iPSCs Induced from BJ FibroblastsUsing RNase III-Treated or HPLC-Purified Unmodified or ψ-Modified mRNAsEncoding iPSC Induction Factors Materials and Methods for Example 25.

iPSCs derived from BJ fibroblasts were maintained in culture originallyon NUFF feeder cells in iPS medium with 10 ng/ml FGFb, then on MATRIGELartificial matrix in mTeSR media as previously described. Threedifferent iPSC lines were differentiated, one from RNAse III-treatedmRNA and two from HPLC-purified mRNA.

Pseudouridine-Modified, RNAse III-Treated mRNA

Line 1 (TN4w4) was derived from BJ cells reprogrammed withpseudoU-modified, RNase III-treated (with 1 mM MgOAc) mRNA of 1:1:1:3:1molar stoichiometric mix of KLM(long)OS. The reprogramming involved 18daily transfections of mRNA into BJ fibroblasts plated on NuFF cells inPluriton media, as previously described. (Note this is the same iPSCline that was examined by qPCR to BJ fibroblasts to compare expressionpatterns.) A colony was picked and expanded for 17 passages, then frozendown for a week, then brought up and passaged 4 more times. Largecolonies were allowed to form, were detached from the matrigel surfacewith dispase, and were kept in suspension culture for 8 days in iPSmedia with no FGFb to allow embryoid body formation. As describedpreviously, the embryoid bodies were then plated on gelatin coatedplates and allowed to attach and spontaneously differentiate in iPSmedia without FGFb for an additional 7 days. The cells were then fixedand incubated with antibodies for various markers as previouslydescribed. Immunofluorescence was performed and the cells were imaged.

Results for Example 25.

The iPSCs stain positively for markers representing all 3 germ layers ofcells. Cells were found that expressed the ectoderm markers, neuronalclass III beta-tubulin (TUJ1), Glial Fibrillary Acidic Protein (GFAP)and neurofilament-light (NF-L), the mesoderm markers, alpha-smoothmuscle actin (SMA) and desmin, and the endoderm markers, transcriptionfactor SOX17 and alpha fetoprotein (AFP). Thus, pseudouridine-modified,RNAse III-treated cap1, poly(A)-tailed, mRNA mixes can be used togenerate iPSCs that differentiated into cells of all 3 germ layers.

Line 2 iPSC Differentiation: Pseudouridine-Modified, HPLC-Purified mRNA

Line 2 (TN8w3) was derived as described above from pseudoU-modified,HPLC-purified, mRNA mixes that contained the shorter cMyc T58A mRNA. Theline has been passaged 11 times before embryoid bodies were formed.

As is shown in FIG. 47, the iPSCs stain positively for markersrepresenting all 3 germ layers of cells. Cells were found that expressedthe ectoderm marker, neuronal class III beta-tubulin (TUJ1), themesoderm markers, alpha-smooth muscle actin (SMA) and desmin, and theendoderm markers, transcription factor SOX17 and alpha fetoprotein(AFP).

Line 3 iPSC Differentiation: Pseudouridine-Modified HPLC-Purified mRNA

Line 3 (TN18w35) was derived (as Line 2 was) from pseudoU-modified,HPLC-purified, mRNA mixes that contained the shorter cMyc T58A mRNA. Theline has been passaged 4 times before embryoid bodies were formed. Thisis a newer line, but was confirmation of reprogramming withHPLC-purified mRNA.

The iPSCs stain positively for markers representing all 3 germ layers ofcells. Cells were found that expressed the ectoderm marker neuronalclass III beta-tubulin (TUJ1), the mesoderm markers alpha-smooth muscleactin (SMA) and desmin, and the endoderm marker SOX17. Results are shownin FIG. 48.

Example 26. Use of Single-Stranded Pseudouridine-Containing mRNAsEncoding iPSC Induction Factors for Feeder-Free Reprogramming of HumanSomatic Cells to iPS Cells on Tissue Culture Plates that were Pre-Coatedwith Vitronectin XF or that were without Coating with Vitronectin or anyOther Extracellular Matrix or Biological Substrate

Pseudouridine-modified RNA encoding SOX2, KLF4, LIN28, OCT4, andcMYC(T58A) reprogramming factors were in vitro-transcribed, treated withRNAse III with 2 mM magnesium acetate, and then enzymatically capped,and poly(A)-tailed, all as previously described.

For feeder-free reprogramming on Vitronectin XF-coated plates, ThermoScientific Nunc Untreated Multidishes (Fisher Scientific, catalog no.12-566-80; Thermo Scientific no. 150239), were coated with VitronectinXF™ (Primorigen Biosciences, Inc. Madison, Wis., USA) according tomanufacturer's instructions and incubated at 37° C. at least 3 hoursbefore plating cells.

For feeder-free reprogramming directly on plates without coating (e.g.,without coating with vitronectin or any other extracellular matrix orbiological substrate), Thermo Scientific Nunc Nunclon delta treatedMultidishes (Fisher Scientific, catalog no. 14-832-11; Thermo Scientificno. 140675) were used; this product is listed as “Nunclon deltatreated,” which the supplier describes as “not coated with any chemicalreagents,” but “a surface modification which enhances cell attachmentand growth for adherent cell lines.”

BJ fibroblasts were plated onto either the Vitronectin XF-coated tissueculture plates or the tissue culture plates without vitronectin or anyother coating at 1×10⁵ or 5×10⁴ cells per well in a minimum essentialmedium (MEM) useful for growth of fibroblast cells comprising: AdvancedMEM (Invitrogen, Carlsbad, Calif., USA) supplemented with 10% FBS(Fisher Scientific), 2 mM GLUTAMAX™-I (Invitrogen) andpenicillin-streptomycin antibiotics, and incubated overnight at 37° C.,5% CO₂.

The following day, the medium was replaced with a Feeder-freeReprogramming Medium developed by the present Applicants consisting ofDulbecco's modified Eagle medium with nutrient mixture F-12 (DMEM/F12;(DMEM/F12; Invitrogen) supplemented with 20% KNOCKOUT™ serum replacement(Invitrogen), 2 mM GLUTAMAX™-I (Invitrogen), 0.1 mM non-essential aminoacids solution (Invitrogen), 8 micromolar transforming growth factor β(TGFβ) inhibitor STEMOLECULE™ SB431542 (Stemgent®, Cambridge, Mass.,USA), 0.5 micromolar MEK signaling pathway inhibitor STEMOLECULE™PD0325901 (Stemgent), and 100 ng/ml basic human recombinant fibroblastgrowth factor (FGFb; Invitrogen) with penicillin-streptomycinantibiotics. Medium was replaced daily prior to transfection ofreprogramming mRNAs using RNAiMAX transfection reagent (Invitrogen). AmRNA/RNAiMAX complex in Opti-MEMI Reduced Serum Medium (Invitrogen) wasprepared separately for each well containing cells to be reprogrammed:briefly, the mRNA reprogramming mix for one well of a plate was added toa first 60-microliter aliquot of Opti-MEMI Reduced Serum Medium; thenthis mRNA-containing first aliquot was combined with a second60-microliter aliquot of Opti-MEMI Reduced Serum Medium containing 5microliters of RNAiMAX transfection reagent per microgram of mRNA added;and finally, this mRNA/RNAiMAX complex in Opti-MEMI Reduced Serum Mediumwas incubated at room temperature for 15 minutes and then added dropwiseto the cells in the well. Once the medium/mRNA/RNAiMAX mixture was addedto all wells, the plates were incubated overnight at 37° C., 5% CO₂. Forfeeder-free reprogramming in Vitronectin XF-coated plates, cells weretransfected in this way for 21 consecutive days. For feeder-freereprogramming of cells in plates that were not pre-coated withvitronectin or another extracellular matrix or other biologicalsubstrate, cells were transfected in this way for 18 consecutive days.Following the last transfection, cells were maintained in iPSCMaintenance Medium until the colonies were large enough to pick.

Several days after the last transfection, the number of colonies thatexhibited the morphology characteristic of iPS colonies were counted inwells of each type of plate using the above treatment protocols (i.e.,in wells of the Vitronectin XF-coated plates and in wells of the plateswhich were not pre-coated with vitronectin or another extracellularmatrix or other biological substrate), and then representative iPSCcolonies from each type of treatment and protocol were manually picked,and grown in half mTESR/half iPSC Maintenance Medium on plates coatedwith Vitronectin XF™ (Primorigen Biosciences, Inc.) to generate iPSCcell lines for further characterization. Putative iPS cell lines werethen transitioned and maintained on Vitronectin XF in mTESR™ medium(Stem Cell Technologies, Vancouver, BC, Canada). Once expanded, theselines were then characterized by staining for pluripotency markers andby using them in the embryoid body spontaneous differentiation protocol,as previously described.

Results for Feeder-Free Reprogramming of BJ Fibroblasts to iPS Cells

Colonies characteristic of iPS cells were visually observed formingafter 17 transfections, both on the plates coated with Vitronectin XFand on the plates which were not pre-coated with vitronectin or anotherextracellular matrix or other biological substrate.

The tables below show the number of iPSC colonies induced in wells foreach type of plate and treatment protocol.

Number of iPSC Colonies Reprogrammed on Feeder-free VitronectinXF-coated Plates Cell mRNA Dose No. of iPSC density Plated (μg/well/day)Colonies Observed 1 × 10⁵ Mock (No RNA) 0 1 × 10⁵ 1.0 31 1 × 10⁵ 1.2 485 × 10⁴ Mock (No RNA) 0 5 × 10⁴ 1.0 17 5 × 10⁴ 1.2 18

Number of iPSC Colonies Induced Directly on Nunc Cell Culture TreatedMultidish Plates That Were Not Coated with an Extracellular Matrix orOther Biological Substrate. Cell mRNA Dose No. of iPSC Density Plated(μg/well/day) Colonies Observed 1 × 10⁵ Mock (No RNA) 0 1 × 10⁵ 0.8 11 1× 10⁵ 1.0 3 1 × 10⁵ 1.2 0 1 × 10⁵ 1.4 1 5 × 10⁴ Mock (No RNA) 0 5 × 10⁴0.8 3 5 × 10⁴ 1.0 1 5 × 10⁴ 1.2 1 5 × 10⁴ 1.4 2

Representative cell lines from both Vitronectin XF-coated plates andfrom plates which were not pre-coated with vitronectin or anotherextracellular matrix or other biological substrate stained positivelyfor the pluripotency markers OCT4, NANOG, TRA-1-60, SSEA4 and SOX2, and,when subjected to the embryoid body spontaneous differentiationprotocol, cells of these cell lines spontaneously differentiated intocells of all 3 germ layers, as shown by positive immunofluorescentstaining for markers specific for cells of each germ layer, includingfor SOX17 (endoderm), DESMIN (mesoderm), and BETA-III tubulin(ectoderm).

As described above, these exemplary experiments further demonstratedembodiments of the present invention, wherein said introducing ofmodified mRNA comprising pseudouridine-containing mRNA encoding iPSCinduction factors induced reprogramming of mammalian cells thatexhibited a first differentiated state or phenotype (in this case,somatic cells comprising human BJ fibroblasts) to cells that exhibited asecond state of differentiation or phenotype (in this case, iPS cells).Still further into this particular embodiment, said reprogramming was inthe absence of any inhibitor or agent that reduces expression oractivity of an innate immune response pathway (e.g., B18R protein wasnot present prior to, during, or after said introducing of thepseudouridine-containing mRNA into said cells).

One other embodiment of the present invention is a Feeder-freeReprogramming Medium consisting of Dulbecco's modified Eagle medium withnutrient mixture F-12 (DMEM/F12; Invitrogen) supplemented with 20%KNOCKOUT™ serum replacement (Invitrogen), 2 mM GLUTAMAX™-I (Invitrogen),0.1 mM non-essential amino acids solution (Invitrogen), and 0.5-15micromolar MEK signaling pathway inhibitor STEMOLECULE™ PD0325901(Stemgent). In some embodiments, the Feeder-free Reprogramming Mediumfurther comprises transforming growth factor β (TGFβ) inhibitorSTEMOLECULE™ SB431542 (Stemgent®, Cambridge, Mass., USA). In someembodiments, the Feeder-free Reprogramming Medium further comprises 100ng/ml basic human recombinant fibroblast growth factor (FGFb;Invitrogen). In some embodiments, the Feeder-free Reprogramming Mediumfurther comprises penicillin and streptomycin antibiotics.

Example 27. Further Studies on the Abilities of Unmodified andPseudouridine-Modified mRNAs Having Different Caps to Reprogram SomaticCells to iPSCs with or without RNase III Treatment or HPLC PurificationMaterials and Methods for Example 27

Synthesis of mRNAs for Reprogramming

The mRNAs referred to only as “CAP0 OR CAP1” without additionaldesignation of a dinucleotide cap analog were synthesized by in vitrotranscription (IVT) of DNA templates encoding the 5 reprogrammingfactors (KLM_(T58A)OS) as described in the T7 mScript™ Standard mRNAProduction System (CELLSCRIPT, INC., Madison, Wis., USA) for unmodified(GAUC) mRNA. Pseudouridine- (ψ-) modified mRNA, was similarlysynthesized by IVT, except with pseudouridine-5′-triphosphate (ψTP) inplace of UTP. The IVT-mRNAs were then post-transcriptionally capped toCAP0 using SCRIPTCAP™ capping enzyme or to CAP1 using SCRIPTCAP™ cappingenzyme and SCRIPTCAP™ RNA 2′-O-methyltransferase, as described in the T7mScript™ Standard mRNA Production System, or as described for theseparate SCRIPTCAP™ capping enzyme and/or SCRIPTCAP™ RNA2′-O-methyltransferase products (CELLSCRIPT, INC.). For mRNAs cappedwith a P-S-ARCA D1 or β-S-ARCA D2 dinucleotide cap analogs, also hereinreferred to specifically as D1 or D2 thio-ARCAs, or generally asthio-ARCAs (Grudzien-Nogalska E et al. 2007; Kowalska, J et al., 2008),the mRNAs were made by co-transcriptional capping by including therespective dinucleotide cap analog in the IVT reaction at a molar ratioof 4-to-1 with GTP, at concentrations as described in a MessageMAX™ T 7ARCA-Capped Message Transcription Kit (CELLSCRIPT, INC. Cat. No.C-MMA60710), except that the respective P-S-ARCA D1 or D2 dinucleotidecap was used in place of the ARCA provided in the kit. All of the mRNAswere enzymatically tailed using A-PLUS™ poly-A polymerase (CELLSCRIPT,INC., Catalog No. C-PAP5104H) to generate a poly-A tail of ˜150 nt, asdescribed by the manufacturer. The mRNAs that were treated using theRNase III treatment method disclosed herein in the presence of 2 mMmagnesium acetate. Certain CAP1 pseudouridine-modified mRNAs were HPLCpurified by Dr. Drew Weissman and Dr. Katalin Karikó of RNARx LLC(Wayne, Pa.) using HPLC as described (Karikó et al., 2011).

Reprogramming with GAUC Unmodified mRNA Mixes

Five-factor mRNA reprogramming mixes (KLM_(T58A)OS) encoding KLF4 (K),LIN28 (L), cMYC(T58A)(MT58A), OCT4(O) and SOX2(S) were made in a molarratio of 1:1:1:3:1, and 1.2 micrograms of each mRNA mix was complexedwith 4.8 microliters of STEMFECT™ transfection reagent (Stemgent) andtransfected daily into 10¹ BJ fibroblasts (passage 5) plated on 4×10⁵NuFF cells. No inhibitor of innate immune response pathway (e.g., B18Rprotein) was used for reprogramming in the experiments reported here. Insome cases, 2 mM valproic acid was added; however, these experimentswill not be discussed further since all of the cells treated withvalproic acid died. Cells were transfected with unmodified GAUC mRNAreprogramming mixes for 18 daily transfections, after which the cellswere grown for 2 more days, a few colonies were picked for expansion andthe rest were stained for alkaline phosphatase activity, which isindicative of iPSCs, and alkaline phosphatase-positive colonies werecounted. Cells reprogrammed using pseudouridine-modified GAψC mRNAreprogramming mixes were transfected for only 15 daily transfections,and in some cases, 1.0, 1.2 or 1.4 micrograms of eachpseudouridine-modified GAψC mRNA reprogramming mix was transfected with4, 4.8 and 5.6 microliters of STEMFECT transfection reagent,respectively. The other steps of the reprogramming method usingpseudouridine-modified GAψC mRNA reprogramming mixes were as describedfor the unmodified GAUC mRNA.

Results for Example 27

Comparison of iPSC Induction Using HPLC-Purified Versus RNaseIII-Treated Pseudouridine-Modified CAP1 KLMO₃S mRNA Reprogramming Mixes

No. of Alkaline Purification or Micrograms of mRNA Phosphatase-Treatment Reprogramming Mix Positive Method Transfected Per DayObservations Colonies None 1.2 CELLS DEAD  0 HPLC 1.0 148  HPLC 1.2TMTCA 400+ HPLC 1.4 TMTCA 400+ RNase III 1.0 149  RNase III 1.2 TMTCA400+ RNase III 1.4 TMTCA 400+ TMTCA = Too many colonies to countaccurately.

The above results show that mRNA reprogramming mixes comprisingpseudouridine-modified mRNAs were highly toxic to cells into which theywere transfected daily for 15 days. However, when the same mRNAreprogramming mixes were purified by HPLC or were treated using theRNase III treatment methods described herein, the cells survived andiPSC cells were induced. The fact that the numbers of alkalinephosphatase-positive colonies, which is indicative of iPS cells, werenearly identical for the wells transfected with HPLC-purified and theRNase III-treated reprogramming mRNAs (e.g., 148 alkalinephosphatase-positive iPSC colonies induced using 1.0 micrograms per wellof reprogramming mix made using HPLC-purified mRNAs versus 149 alkalinephosphatase-positive iPSC colonies induced using 1.0 micrograms per wellof reprogramming mix made using the same lots of mRNAs that were treatedusing the RNase III treatment methods described herein) stronglyindicates that dsRNA the primary RNA contaminant that results in celldeath and the inability to reprogram somatic cells using mRNAreprogramming mixes comprising mRNAs which have not been HPLC-purifiedor RNase III-treated. In view of the equivalent effectiveness of theRNase III treatment methods described herein to HPLC purification inremoving dsRNA contaminant molecules from mRNA, other important benefitsof the present RNase III treatment method make it advantageous over HPLCpurification.

Reprogramming of Somatic Cells to iPS Cells Using In Vitro-SynthesizedUnmodified GAUC mRNAs Encoding KLMO₃S Reprogramming Factors andComprising Co-Transcriptionally-Synthesized Thio Caps or EnzymaticallyPost-Transcriptionally Synthesized Cap0 or Cap1 Caps

No. of Alkaline Subjected to Phosphatase- NTP the RNase III Positive CAPType mix Treatment Observations Colonies No RNA None NO No significant 0Control toxicity β-S-ARCA GAUC NO Cells died 0 D1 β-S-ARCA GAUC NO Cellsdied 0 D2 CAP0 GAUC YES 4 CAP1 GAUC YES 289

The results above showed that mRNA reprogramming mixes comprisingunmodified mRNAs were highly toxic to cells into which they weretransfected daily for 18 days. However, when the same mRNA reprogrammingmixes were treated using RNase III treatment with 2 mM Mg²⁺, the cellssurvived and iPSC cells were induced. The results further showed thatmRNA reprogramming mixes comprising unmodified GAUC mRNAs that exhibiteda CAP1 structure were much more effective for reprogramming somaticcells to iPS cells than unmodified (GAUC) mRNAs that exhibited a CAP1structure. Thus, in preferred embodiments of the reprogramming methods,compositions and kits of the invention comprising mRNA reprogrammingmixes comprising unmodified mRNAs, the unmodified mRNAs exhibit a CAP1structure.

Example 28. Effects of Pseudouridine-Modified (GAψC) or Unmodified(GAUC) dsRNA on Reprogramming of Human BJ Fibroblasts to iPS Cells UsingRNase III-Treated Cap1 Poly(A)-Tailed GAψC of GAUC mRNAs Encoding KLMO₃SReprogramming Factors Overview

Previous results, including those discussed in EXAMPLE 27, showed theequivalence of the RNase III treatment methods (e.g., using about 2 mMMg²⁺) to HPLC for removing contaminant dsRNA from mRNA reprogrammingmixes (e.g., 148 alkaline phosphatase-positive iPSC colonies wereinduced using 1.0 micrograms per well of reprogramming mix made usingHPLC-purified pseudouridine-modified CAP1 KLM_((T58A))O₃S mRNAs versus149 alkaline phosphatase-positive iPSC colonies induced using 1.0micrograms per well of a reprogramming mix made using the same lots ofmRNAs that were treated using the RNase III treatment methods describedherein). Since approximately all of the dsRNA contaminants are removedusing these methods, the present researchers saw this as an opportunityto analyze the levels of dsRNA contaminant that would result in toxicityand that would reduce or inhibit reprogramming, such as reprogramming ofhuman or mammalian somatic cells to iPS cells, by adding back differentknown amounts of dsRNA to the mRNA reprogramming mixes. In order toavoid a biological effect (e.g., a biological effect due to RNAinterference), the dsRNA chosen to add to the mRNA reprogramming mixeswas a dsRNA made using a DNA template that was not present in the cellsinto which the mRNA reprogramming mixes were introduced; a 1.67-Kbfirefly luciferase gene (luc2), which did not appear to be present inhuman cells, was chosen as the template for making dsRNA for thispurpose. After making luc2 dsRNA by IVT, various amounts of the 1.6-Kbluc2 dsRNA were added to mRNA reprogramming mixes comprising mRNAs thatwere treated using the RNase III treatment method with 2 mM Mg2+ beforethe transfection reagent was added; in separate wells of 6-well plates,pseudouridine-modified (GAψC) or unmodified (GAUC) luc2 dsRNA was addedto a reprogramming mix comprising either RNase III-treatedpseudouridine-modified (GAψC) CAP1 KLM_((T58A))O₃S mRNAs or RNaseIII-treated unmodified (GAUC) CAP1 KLM_((T58A))O₃S mRNAs in order to tryto tease out any differences between the cells' reaction to dsRNA andthe cells' reaction to pseudouridine-modified versus unmodified mRNA.

Summary of the Protocol

Except for controls, all mRNAs in mRNA reprogramming mixes were treatedusing the RNAse III treatment method with 2 mM Mg2+.

All mRNAs were post-transcriptionally capped using SCRIPTCAP™ cappingenzyme system and SCRIPTCAP™ RNA 2′-O-methyltransferase to CAP1.

All mRNAs were poly-A tailed to ˜150 As using A-PLUS™ poly-A polymerase.

Two different mRNA reprogramming mixes: one comprising unmodified (GAUC)mRNAs and one comprising pseudouridine-modified (GAψC) mRNAs.

5-Factor mRNA reprogramming mixes encoding KLM_((T58A))OS in a molarratio of 1:1:1:3:1 were used; K=KLF4; L=LIN28; M_((T58A))=cMYC(T58A);O═OCT4; S═SOX2.

Cells were transfected with mRNA reprogramming mixes were transfecteddaily with a total of 1.2 micrograms of mRNA encoding all 5 proteinfactors (KLM_((T58A))OS) per well for 14 days for GAψC mRNAs or for 18days for GAUC mRNAs. The indicated amounts of luc2 dsRNA was combinedwith the mRNA reprogramming mix prior to complexing with the STEMFECT™transfection reagent, and then added the mRNA/dsRNA/transfection reagentcomplex was added to the medium as described.

Materials and Methods for Example 28

Synthesis of luc2 dsRNA

Both pseudouridine-modified GAψC dsRNA and unmodified GAUC dsRNAscomprising sense and antisense ssRNA for a genetically engineered formof the firefly (Photinus pyralis) luciferase gene designated “luc2”(˜1.67 Kbp) were produced as follows: Two linear DNA templates forseparate in vitro transcription of sense and antisense ssRNAs weregenerated by restriction endonuclease linearization of a pGL4.19[luc2-Neo] plasmid (Promega, Madison, Wis., USA) that was modified byPCR to insert T7 and T3 RNA polymerase promoters, respectively. Eachsense or antisense ssRNA strand was synthesized separately by in vitrotranscription of a linear luc2 DNA template using either 17 RNApolymerase or T3 RNA polymerase, such as with a commercially availableINCOGNITO™ T7 Ψ-RNA transcription kit (CELLSCRIPT, INC., Madison, Wis.,USA) for making GAψC RNA, or a T7-FLASHSCRIBE™ transcription kit or aT7-SCRIBE™ standard RNA IVT kit for making GAUC RNA (CELLSCRIPT), orsimilar home-brew kits containing T3 RNA polymerase. The firefly luc2dsRNA was not capped or tailed. Each sense or antisense ssRNA was thenseparately resuspended in T₁₀E1, combined in equal amounts, annealed at94° C. for 2 minutes, 70° C. for 10 minutes, and then slow-cooled toroom temperature in a beaker of water. Fresh dsRNA dilutions were madedaily in water because of the extremely low amounts of luc2 dsRNA addedto the mRNA reprogramming mixes. The amount of luc2 dsRNA added for eachdaily treatment and the dsRNA added as a percentage of the total amountof RNA transfected per day are listed in the table below.

The Reprogramming Protocol

As in previous experiments, 10⁴ BJ fibroblasts (passage 6) were platedon NUFFs and the medium was changed to Stemgent's PLURITON medium (withsupplement and pen/strep) with RNase Inhibitor added to 0.5U/ml ofmedium. The STEMFECT transfection reagent was used as previouslydescribed. Briefly, 1.2 micrograms of the appropriate mRNA reprogrammingmix was added to STEMFECT buffer with varied amounts of eitherpseudouridine-modified or unmodified dsRNA. The STEMFECT transfectionreagent was separately diluted in STEMFECT transfection buffer the twomixes were combined and incubated at RT for 15 minutes. The mixture wasthen added drop-wise to the cells which were in 2 mls of PLURITONreprogramming medium/well. The medium was changed daily prior totransfection. The cells transfected with pseudouridine-modified mRNAreprogramming mixes were transfected daily for a total of 14 times. Thecells transfected with unmodified GAUC mRNA reprogramming mixes weretransfected 18 times. Observations on cell health and morphology weremade for the duration of the 20-day experiment. The cells were allowedto form iPSC colonies for 1-2 days after the transfections before thecells were scored for proper colony morphology and stained forenumeration of alkaline phosphatase-positive iPSC colonies.

Overview of Experiment and Final Alkaline Phosphatase-Positive iPSCColony Count

dsRNA as Final No. of % of total FF Luc2 Amount of Amount of AlkalineRNA dsRNA Type/ Reprogramming dsRNA per Phosphatase- Well transfectedReprogramming mRNA per well well positive No. (%) mRNA Type*(micrograms) (nanograms) Colonies⁺  1 0 None/None 0 0 0  6 0 None/U   1.2 0 234  7/8 2.5 U/U 1.2 31 0  9/10 0.5 U/U 1.2 6 0 11/12 0.1 U/U 1.21.2 0 13/14 0.05 U/U 1.2 0.6 0 15/16 0.02 U/U 1.2 0.24 0 17/18 0.01 U/U1.2 0.12 0 19/20 0.008 U/U 1.2 0.096 0 21/22 0.004 U/U 1.2 0.048 0 23/240.0008 U/U 1.2 .0096 1/0 25/26 0.00016 U/U 1.2 .00192 50/38 27 0None/Ψ   1.2 0 400+  28 2.5 Ψ/Ψ 1.2 30 0 29 0.5 Ψ/Ψ 1.2 6 0 30 0.1 Ψ/Ψ1.2 1.2 0 31 0.05 Ψ/Ψ 1.2 0.6 0 32 0.02 Ψ/Ψ 1.2 0.24 0 33 0.01 Ψ/Ψ 1.20.12 0 34 0.008 Ψ/Ψ 1.2 0.096 2 35 0.004 Ψ/Ψ 1.2 0.048 5 36 0.0008 Ψ/Ψ1.2 .0096 400+  37 0.00016 Ψ/Ψ 1.2 .00192 400+  38 0 None/Ψ   1.2 0400+  39 2.5 U/Ψ 1.2 30 0 40 0.5 U/Ψ 1.2 6 0 41 0.1 U/Ψ 1.2 1.2 0 420.05 U/Ψ 1.2 0.6 0 43 0.02 U/Ψ 1.2 0.24 0 44 0.01 U/Ψ 1.2 0.12 0 450.008 U/Ψ 1.2 0.096 0 46 0.004 U/Ψ 1.2 0.048 0 47 0.0008 U/Ψ 1.2 .009615  48 0.00016 U/Ψ 1.2 .00192 400+  2/3 0.1    U/None 0 1.2 0 4/5 0.004  Ψ/None 0 0.048 0 *U = unmodified GAUC RNA, Ψ = pseudouridine-modifiedGAΨC RNA; ⁺400+ = there are more than 400 colonies, too many to countaccurately

Results and Observations for Example 28 BACKGROUND AND INTRODUCTION

We determined in previous experiments (e.g., as in other above Examples)that no alkaline phosphatase-positive iPSC colonies were induced when byBJ fibroblasts or keratinocytes were repeatedly transfected with mRNAreprogramming mixes comprising CAP1 pseudouridine-modified GAΨC mRNAs orGAUC miRNAs encoding KLM_((T58A))O₃S unless the dsRNA contaminantsarising during in vitro transcription were removed using a method suchas HPLC or the RNase III treatment method as described herein.

Still further, we demonstrated in EXAMPLE 27 that mRNA reprogrammingmixes comprising pseudouridine-modified CAP1 mRNAs that were treatedwith the RNase III treatment method with 2 mM Mg²⁺, as described herein,resulted in reprogramming of almost the same number of BJ fibroblasts toalkaline phosphatase-positive iPSC colonies as did the same quantity ofthe same mRNA reprogramming mix comprising the same mRNAs except thatthey were purified using HPLC. This showed that dsRNA was the maincontaminant that inhibited reprogramming and that the RNase IIItreatment methods described herein were as effective as HPLC in removingthe dsRNA contaminant molecules. Therefore, all mRNA reprogramming mixesencoding KLM_((T58A))O₃S used in this EXAMPLE 28, including both thosecomprising CAP1 pseudouridine-modified GAΨC mRNAs and those comprisingCAP1 unmodified GAUC mRNAs, were treated using RNase III treatment inthe presence of 2 mM of Mg²⁺ as described herein.

In the Absence of Added dsRNA, RNase III-Treated GAΨC or GAUCReprogramming mRNA Mixes Efficiently Reprogrammed BJ Fibroblasts to iPSCells.

As shown in the above table, all of the mRNA reprogramming mixescomprising mRNAs that were treated with the RNase III induced largenumbers of alkaline phosphatase-positive iPSC colonies when no dsRNA wasadded to the mRNA reprogramming mixes. Thus, GAΨC reprogramming mRNAsinduced >400 iPSC colonies per well (wells 27 & 38), which was “toonumerous to count accurately,” and the GAUC reprogramming mRNAs induced234 iPSC colonies (well 6). As found in previous experiments, the numberof iPSC colonies induced by unmodified GAUC reprogramming mRNAs was onlyabout half of the numbers and took longer to form colonies compared tothose induced by the modified GAΨC reprogramming mRNAs. No colonies wereinduced in control wells that lacked any reprogramming mRNAs (wells1-5).

Addition of dsRNA to RNase III-Treated GAΨC or GAUC Reprogramming mRNAMixes Increased Cell Toxicity and Decreased iPSC ReprogrammingEfficiency.

The Applicants were surprised by the unexpectedly low levels of dsRNAthat were toxic for the BJ fibroblasts and feeder cells and by the evenlower levels of dsRNA that were required in order to successfullyreprogram the BJ fibroblasts to iPS cells.

For example, with respect to toxicity, we found that addition of dsRNAto the mRNA reprogramming mixes to a level of 0.01% or more of the totalmass of RNA added was toxic to the cells, whether the dsRNA or the mRNAreprogramming mixes, or both, comprised modified GAΨC RNA or unmodifiedGAUC RNA. Thus, all of the cells were dead by day 6 of the treatments ifmore than 1 ng of dsRNA was added with the 1.2-micrograms-per-well perday of mRNA reprogramming mix (i.e., wherein the dsRNA was 0.1% or moreof the total RNA added). All of the cells were dead by the day 10 ifmore than 240 pg of dsRNA was added with the 1.2-micrograms-per-well perday of mRNA reprogramming mix (i.e., wherein the dsRNA was 0.02% or moreof the total mass of RNA added per well). Still further, the cells weredead by the 13^(th) transfection if more than 120 pg of dsRNA was addedwith the 1.2-micrograms-per-well per day of mRNA reprogramming mix(wherein the dsRNA was 0.01% or more of the total mass of RNA added perwell).

Surprisingly and unexpectedly, it was necessary to reduce the level ofdsRNA added to the mRNA reprogramming mix much more still in order tosuccessfully reprogram the BJ fibroblasts to iPS cells during the14-to-18-day iPSC reprogramming protocol.

For example in some embodiments of the method for reprogramming of thepresent invention wherein an mRNA reprogramming mix comprising RNaseIII-treated pseudouridine-modified GAΨC mRNAs encoding one or morereprogramming factors are repeatedly or continuously introduced (e.g.,transfected) into a cell that exhibits a first state of differentiation(e.g., a somatic cell; e.g., a fibroblast or keratinocyte) underconditions wherein the cell exhibits a second state of differentiation(e.g., a dedifferentiated state, a transdifferentiated state, or adifferentiated state; e.g., an iPSC state of differentiation), theamount of dsRNA contaminant molecules in the mRNA reprogramming mix usedfor said introducing into the cell that exhibits the first state ofdifferentiation is less than about 0.01% (and preferably less than about0.001%) of the total RNA used for said introducing. For example, whenthe BJ fibroblast cells were transfected with pseudouridine-modifiedGAΨC dsRNA added to an mRNA reprogramming mix comprising RNaseIII-treated pseudouridine-modified GAΨC mRNAs, iPSCs were not induceduntil the amount of dsRNA was 0.008% or less of the total mass of RNAper well. Even at that level of dsRNA, only 2 iPSC colonies were induced(well 34) in the presence of 96 pg of dsRNA added with the1.2-micrograms-per-well per day of mRNA reprogramming mix (i.e., whereinthe dsRNA was 0.008% of the total mass of RNA added per well). Stillfurther, only 5 iPSC colonies were induced (well 35) in the presence of48 pg of dsRNA added with the 1.2-micrograms-per-well per day of mRNAreprogramming mix (i.e., wherein the dsRNA was 0.004% of the total massof RNA added per well). When 1.92 pg of GAΨC dsRNA was added with the1.2-micrograms-per-well per day of mRNA reprogramming mix (wherein thedsRNA was 0.0008% of the total mass of RNA added per well), noinhibition of iPSC induction was observed for the mRNA reprogramming mixcomprising modified GAΨC mRNAs (well 36).

Only 1 iPSC colony was induced in one of two replicate wells (wells 23and 24) transfected with 9.6 pg of dsRNA added with the1.2-micrograms-per-well per day of mRNA reprogramming mix (i.e., whereinthe dsRNA was 0.0008% of the total mass of RNA added per well). MoreiPSC colonies were induced (50 & 38 colonies in replicate wells 25 and26) with only 1.92 pg of dsRNA added with the 1.2-micrograms-per-wellper day of mRNA reprogramming mix (i.e., wherein the dsRNA was only0.00016% of the total mass of RNA added per well), but even this smallamount of the ˜1.67 Kbp dsRNA decreased the number of viablereprogrammed iPSC colonies by about 80% compared to the number of iPSCcolonies induced when no dsRNA was added to the mRNA reprogramming mix(234 colonies).

In one additional set of experiments, BJ fibroblast cells weretransfected with unmodified GAUC dsRNA and an mRNA reprogramming mixcomprising RNase III-treated pseudouridine-modified GAΨC mRNAs; it willbe recognized that this is an artificial situation that is unlikely tooccur, since dsRNA contaminant molecules are generated during in vitrotranscription reactions and will comprise modified or unmodified RNAbased on whatever NTPs are used in the IVT reaction. Therefore, thedsRNA will not comprise unmodified RNA and the mRNA made by IVT modifiedRNA. Nevertheless, in this set of experiments BJ fibroblast cells weretransfected with unmodified GAUC dsRNA and an mRNA reprogramming mixcomprising RNase III-treated pseudouridine-modified GAΨC mRNAs in orderto determine if dsRNA comprising GAΨC RNA had a different effect on celltoxicity and reprogramming of somatic cells to iPS cells than dsRNAcomprising GAUC RNA. Thus, the highest dose of dsRNA at which iPSCcolonies were induced was at 9.6 pg of dsRNA added with the1.2-micrograms-per-well per day of mRNA reprogramming mix (wherein thedsRNA was 0.0008% of the total mass of RNA added per well); at thisdose, 15 iPSC colonies were induced (well 47). The cells tolerate thisreprogramming mix better though. The cells were healthier and morecolonies were obtained at the lowest dose of dsRNA. This amount ofunmodified GAUC dsRNA was similar to the highest dose of unmodified GAUCdsRNA at which iPSC colonies were induced when an mRNA reprogramming mixcomprising unmodified GAUC mRNAs was used (wells 23 and 24). However, inthe presence of unmodified GAUC dsRNA, the use of an mRNA reprogrammingmix comprising GAΨC mRNAs did seem to reduce cell toxicity and increasereprogramming efficiency compared to the use of an mRNA reprogrammingmix comprising GAUC mRNAs (e.g., compare well 47 with wells 23 and 24,and well 48 with wells 25 and 26). The results with GAΨC dsRNA with anmRNA reprogramming mix comprising GAΨC mRNA (e.g., wells 35, 36 and 37)further demonstrates the benefits of reduced toxicity and increasedreprogramming efficiency by using pseudouridine-modified mRNAs in themRNA reprogramming mixes.

Example 29. Effects of Adding Unmodified (GAUC), Pseudouridine-Modified(GAψC), or Pseudouridine- and 5-Methylcytidine-Modified (GAψm⁵C) Luc2dsRNA on Reprogramming of Mouse C3H/10T1/2 Cells to Myoblast Cells Usinga Reprogramming Mix Comprising RNase III-Treated Cap1, Poly(A)-TailedGAUC, GAψC OR GAψm⁵C mRNA Encoding MYOD Protein

Synthesis of Double-stranded Luciferase2 RNA (luc2 dsRNA)

Linear DNA templates encoding a genetically engineered form of thefirefly (Photinus pyralis) luciferase gene, designated “luc2,” were usedto generate sense and antisense ssRNAs as described in EXAMPLE 28,except that either GAUC or GAψC or GAψm⁵C NTP mixes were used for invitro transcription of both the sense and antisense ssRNAs. The senseand antisense ssRNAs were then separately resuspended in water, combinedin equal amounts, and annealed to generate luc2 dsRNAs comprising GAUCor GAψC or GAψm⁵C nucleotides using the following protocol: 250microliters each of sense and antisense luc2 ssRNAs (each at 1microgram/ml) comprising the same nucleotide composition were addedtogether and heated at 950C for 2 minutes, followed by 70° C. (5minutes), 60° C. (10 minutes), 50° C. (10 minutes), 40° C. (10 minutes),30° C. (10 minutes) and then allowed to cool to room temperature for 30minutes. The GAUC, GAψC and GAψm⁵C dsRNA products were all confirmed tobe double-stranded.

Synthesis of mRNA Encoding MYOD

A mouse MYOD DNA template for preparing mouse mRNA comprising orconsisting of unmodified mouse MYOD mRNA (GAUC) for use in reprogrammingmouse mesenchymal stem cells to myoblast cells was prepared as follows:DNA encoding MYOD mRNA, which mRNA exhibited the coding sequence or cdsgiven as SEQ ID NO: 16, was cloned into pUC19-based plasmid DNA thatcontained a cassette exhibiting SEQ ID NO: 1, comprising a T7 RNApolymerase promoter followed by 5′ Xenopus Beta Globin (UTR), a cloningsite (into which the MYOD cds was inserted directly downstream of aKozak translational initiation site GCCACC), and a 3′ Xenopus BetaGlobin 3′ UTR. The DNA plasmid was linearized with Sal I and purified aspreviously described for other DNA plasmids as described herein, andthen used as a DNA template for in vitro transcription of mRNA encodingMYOD (or MYOD mRNA).

Synthesis of MYOD mRNAs for Reprogramming

CAP1, poly(A)-tailed (˜150 nts) unmodified (GAUC) mRNA encoding the MYODprotein, as encoded by the above-described MYOD DNA template, wassynthesized by in vitro transcription (IVT) of said DNA template asusing the T7 mScript™ Standard mRNA Production System (CELLSCRIPT, INC.,Madison, Wis., USA) as described by the manufacturer. CAP1,poly(A)-tailed (˜150 nts) pseudouridine-modified (GAψC) mRNA andpseudouridine- and 5-methylcytidine-modified (GAψm⁵C) mRNAs were eachsimilarly synthesized by IVT using a T7 mScript™ Standard mRNAProduction System, except that NTP mixes comprising GAψC NTPs or GAψm⁵CNTPs, respectively, were used in place of UTP or CTP. Portions of eachof these unmodified (GAUC) and modified (GAψC and GAψm⁵C) mRNAs weretreated using RNase III treatment in the presence of 2 mM magnesiumacetate as disclosed herein.

Reprogramming of Mouse C3H10T1/2 Mesenchymal Stem Cells to MyoblastCells Using CAP1 Unmodified MYOD mRNA and Effect of Luc2 dsRNA

Mouse C3H10T1/2 cells were plated at 2×10⁵ cells per well of agelatin-coated 6-well dish and grown overnight in DMEM, 10% FBS,GLUTAMAX, and pen/strep. The next day, the cells were switched todifferentiation medium comprising DMEM+2% horse serum, GLUTAMAX, andpen/strep. Cells were transfected using RNAiMAX transfection reagent(Invitrogen, Inc.) with 1.0 micrograms/ml of the above-describedunmodified (GAUC) mRNA or GAψC modified mRNA or GAψm⁵C modified mRNAencoding MYOD protein, either alone with no luc2 dsRNA, or together withluc2 dsRNA comprising the same type of nucleotides (GAUC, GAψC orGAψm⁵C) as the mRNA encoding MYOD protein, with each respective luc2dsRNA in varying concentrations between 0.000001 and 0.1 micrograms/ml.Briefly, each GAUC, GAψC or GAψm⁵C MYOD mRNA and the corresponding luc2dsRNA were added to a first tube containing a total volume of 60microliters and an amount of RNAiMAX transfection solution equal to 5microliters per microgram of RNA in the first tube was added to a secondtube and the final volume was adjusted to 60 microliters. The first andsecond tubes were mixed, incubated at room temperature for 15 minutes,and the mRNA/RNAiMAX mix was added to 2 mls of differentiation mediumalready on the cells. The medium was changed with new differentiationmedium 4 hours post transfection. Twenty-four hours after the firsttransfection, another transfection with the same treatment wasadministered. The medium was again changed 4 hours post transfection.Forty-eight hours after the first transfection, the cells were fixed andimmunofluorescence was performed to detect Myosin Heavy Chain (MHC)expression. a marker of myoblast or muscle differentiation.

Results of Example 29

The percentage of contaminant dsRNA must be less than about 0.1% (andpreferably less than about 0.01%) of the total amount of RNA toreprogram mesenchymal stem cells to myoblast cells using unmodified MYODmRNA or GAψC-modified MYOD mRNA.

Amount of Amount of dsRNA as RNase III- Respective % of Presence ofTreated GAUC or GAψC* Total Myosin GAUC or GAψC Luc2 dsRNA RNA HeavyChain MYOD mRNA Transfected Trans- Immunofluorescent (μg/ml) (μg/ml)fected Staining 1.0 0 0 YES 1.0 0.1    10% No 1.0 0.01    1% No 1.00.001   0.1% No 1.0 0.0001  0.01% YES 1.0 0.00001  0.001% YES 1.00.000001 0.0001% YES Untreated Untreated N/A No Mock Transfected MockTransfected N/A No *GAUC luc2 dsRNA is used with GAUC MYOD mRNA and GAψCluc2 dsRNA is used with GAψC MYOD mRNA. N/A = Not Applicable.The percentage of contaminant dsRNA must be less than 1% (and preferably0.1% or less) of the total amount of RNA to reprogram mesenchymal stemcells to myoblast cells using GAψm⁵C-modified MYOD mRNA.

dsRNA as Amount of Amount of % of Presence of RNase III- GAψm⁵C TotalMyosin Treated GAψm⁵C Luc2 dsRNA RNA Heavy Chain MYOD mRNA TransfectedTrans- Immunofluorescent (μg/ml) (μg/ml) fected Staining 1.0 0 0 YES 1.00.1    10% No 1.0 0.01    1% No 1.0 0.001   0.1% YES 1.0 0.0001  0.01%YES 1.0 0.00001  0.001% YES 1.0 0.000001 0.0001% YES Untreated UntreatedN/A No Mock Transfected Mock Transfected N/A No

Example 30. Direct Reprogramming of Human Fibroblasts to Neurons byRepeated Introduction of Pseudouridine-Modified (GAΨC) mRNAs EncodingASCL1, MYT1L, NEUROD1 and POU3F2 Protein Transcription FactorsINTRODUCTION

Recently, Pang et al. and others (Pang, Z P et al., 2011; Ladewig J, etal. 2012) described the conversion of human fibroblasts to neurons bythe introduction of doxycycline-inducible lentiviral vectors encodingfour transcription factors (ASCL1, MYT1L, NEUROD1 and POU3F2), buildingon work of other researchers (e.g., Vierbuchen T, et al. 2010; Yang N,et al. 2011). In this Example, we show highly efficient directreprogramming (e.g., transdifferentiation) of human fibroblasts toneurons by repeatedly introducing into the fibroblast cells areprogramming mix comprising pseudouridine-modified mRNAs encodingprotein transcription factors (e.g., ASCL1, MYT1L, NEUROD1 and POU3F2),wherein the mRNAs were treated using the RNase III treatment method with2 mM Mg²⁺, thereby reprogramming the fibroblasts to neural cells.

Materials and Methods for Reprogramming Fibroblasts to Neurons Example30 Details

IMR90 fetal human lung fibroblasts (passage P15) were seeded ongelatin-coated plates at 1.5×10⁵ cells per well of a 6-well plate inEMEM (ATCC Cat. No. 30-2003) medium supplemented with 10% fetal bovineserum and 1× penicillin-streptomycin. The cells in each well weretransfected daily (e.g., in this example, for 6 days) with areprogramming mix comprising a total of 0.6 microgram of RNaseIII-treated (with 2 mM Mg²⁺), pseudouridine-modified (GAΨC) recombinantmRNAs (encoding each of ASCL1 (A), MYT1L (M), NEUROD1 (N) and POU3F2 (P)protein transcription factors in a 1:1:1:1 molar ratio of AMNP)complexed with the STEMFECT™ transfection reagent (4 microliters permicrogram mRNA). The recombinant mRNAs were made by in vitrotranscription of linearized pUC19-derived DNA templates that contained acassette (SEQ ID No: 1) comprising: a T7 promoter, a 5′ UTR of Xenopuslaevis β-globin, and a 3′ UTR of Xenopus laevis β-globin; into which aDNA sequence encoding mRNA, which mRNA exhibits a coding sequence asgiven in the following SEQ ID No: ASCL1 (SEQ ID No: 11), MYT1L (SEQ IDNo: 12), NEUROD1 (SEQ ID No: 13), or POU3F2 (SEQ ID No: 14 or SEQ ID No:15) protein was inserted. Recombinant CAP1 mRNAs (with an ˜150 nt polyAtail) encoding ASCL1, NEUROD1 and POU3F2 were prepared as described inthe literature provided with the T7 mSCRIPT™ standard mRNA productionsystem (CELLSCRIPT, INC., Madison, Wis., USA), except that pseudouridine5′ triphosphate (ΨTP) was substituted for uridine 5′ triphosphate (UTP)for IVT, and, prior to capping or polyadenylation, the invitro-transcribed RNAs were treated using RNase III treatment asdescribed herein with a concentration of 2 mM magnesium acetate.Recombinant MYT1L mRNA encoding MYT1L (with an ˜150 nt polyA tail) wasprepared as described in the literature provided with the MessageMAX™ T7 ARCA-Capped Message Transcription Kit (CELLSCRIPT), except with ψTP inplace of UTP during IVT and, prior to polyadenylation with A-PLUS™ polyApolymerase (CELLSCRIPT), the in vitro-transcribed RNA was treated usingRNase III treatment, as described herein, with 2 mM magnesium acetate;this mRNA it was not phosphatase-treated. The cells were kept in EMEMmedium for the first 2 days of transfections then changed to N3 mediumfor the remainder of the experiment. N3 medium (Wemig M, et al. 2002) isDMEM/F12 medium (Life Technologies) supplemented with 25 micrograms permilliliter insulin, 50 micrograms per milliliter transferrin, 30nanomolar sodium selenite, 20 nanomolar progesterone, 100 nanomolarputrescine (all from SIGMA) and supplemented with fresh FGFb daily to 10nanograms per milliliter (R&D Systems) and 1× penicillin-streptomycin.The medium was changed daily before transfection and was supplementedwith 0.5 U/ml of SCRIPTGUARD™ RNase inhibitor (CELLSCRIPT). The AMNPmRNA mix: STEMFECT™ transfection reagent complex were made as permanufacturer's protocol (STEMGENT, Cambridge, Mass., USA), incubated 15minutes at room temperature, and added to the cells. Phase contrastimages of the cells were taken on day 6 and the cells were fixed on day7 and immunofluorescently stained for the presence of the neuronalmarker microtubule-associated protein-2 (MAP2). This is a microtubuleassembly protein that is thought to play an essential role inneurogenesis.

Results for Reprogramming Fibroblasts to Neurons

By the 6^(th) transfection the morphology of most of the cells in theAMNP (ASCL1, MYT1L, NEUROD1, and POU3F2)-transfected wells haddramatically changed to a morphology (FIG. 50 and FIG. 51) andimmunofluorescent staining of the cells was positive for MAP2, whichshowed that the fibroblasts had been reprogrammed (transdifferentiateddirectly to neurons. This reprogramming process was rapid and highlyefficient.

Studies on the Effects of Adding Luc2 dsRNA on Reprogramming ofFibroblasts to Neurons

Materials and Methods

In another experiment, various amounts of either unmodified GAUC luc2dsRNA or modified GAψC luc2 dsRNA were added to reprogramming mixescomprising RNase III-treated GAψC-mRNAs encoding ASCL1, MYT1L, NEUROD1,and POU3F2 (AMNP) to determine and quantify the effects of unmodifiedand ψ-modified dsRNA on reprogramming (transdifferentiation) offibroblasts to neurons. As in similar experiments in all previousEXAMPLES, luc2 dsRNA was used because, since it is not naturally presentin human cells, it was believed that it would not cause a biological orbiochemical effect (e.g., due to RNA interference) as might occur if adsRNA was used which exhibited a sequence encoded by a gene that waspresent in the cells.

As described above for EXAMPLE 30 Details, IMR90 fetal lung fibroblasts(P16) were seeded on gelatin-coated plates at 1.5×10⁵ cells per well ofa 6-well plate in EMEM media.

Cells in each well were transfected daily with an mRNA reprogramming mixcomprising a total of 600 nanograms of RNase III-treatedpseudouridine-modified mRNAs encoding ASCL1, MYT1L, NEUROD1, and POU3F2plus or minus various amounts of either unmodified GAUC orpseudouridine-modified (GAψC−) luc2 dsRNA, all complexed with theSTEMFECT transfection reagent (4 microliter/microgram mRNA), for 4 days.The luc2 dsRNAs were added to mRNA reprogramming mixes as in previousexperiments to determine and quantify the effects of dsRNA onreprogramming fibroblasts to iPSCs (e.g., see EXAMPLE 28). All of themRNAs were pseudouridine-modified and RNAse III-treated. All hadCAP1-caps added enzymatically except for MYT1L, which wasco-transcriptionally capped with ARCA, and all were enzymaticallypolyadenylated to generate a poly(A) tail with ˜150 A residues. Thecells were kept in EMEM medium for the first transfection, then changedto N3 medium for the remainder of the experiment. The medium was changeddaily before transfection and was supplemented with 0.5 U/ml ofSCRIPTGUARD RNase Inhibitor.

Results of Studies on the Effects of Adding Luc2 dsRNA on Reprogrammingof Fibroblasts to Neurons

After the 4^(th) transfection, some cells in wells transfected withmRNAs encoding AMNP had changed morphology. Images were taken of thecells on day 5. Transfections were stopped and the cells were culturedfor an additional 5 days to allow the neurons to mature. Then, the cellswere immunostained to detect expression of neuronal markers, includingMAP2 and NeuN, and the numbers of neurons in each well based onmorphology and immunostaining were counted. Neurons were induced in theabsence of added Luc2 dsRNA and in the presence of certain levels ofadded Luc2 dsRNA. When unmodified GAUC Luc2 dsRNA was added daily withthe GAψC-mRNAs encoding ASCL1, MYT1L, NEUROD1, and POU3F2 (AMNP)reprogramming factors, neurons were induced only if the amount of addedunmodified GAψC Luc2 dsRNA was less than about 0.01% of the total massof RNA used for reprogramming, and significant numbers of neurons weregenerated only if the amount of added unmodified GAUC Luc2 dsRNA wasless than about 0.001% of the total mass of RNA used for reprogramming.When modified GAψC Luc2 dsRNA was added daily with the GAψC-mRNAsencoding AMNP reprogramming factors, neurons were induced only ifpseudouridine-modified GAψC Luc2 dsRNA was less than about 0.02% of thetotal mass of RNA used for reprogramming, and significant numbers ofneurons were generated only if the amount of added unmodified GAUC Luc2dsRNA was less than about 0.004% of the total mass of RNA used forreprogramming.

REFERENCES

Each and all of the following are incorporated herein by reference.

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Nature Immunol. 12: 137-143.-   Product literature, including all descriptions and protocols    therein, for the A-PLUS™ poly(A) polymerase tailing kit, the    AMPLICAP-MAX™ T7 high yield message maker kit, the AMPLICAP™ SP6    high yield message maker kit, Anti-reverse cap analog (ARCA), the    INCOGNITO™ SP6 Ψ-RNA transcription kit, INCOGNITO™ T7 ARCA 5mC- &    Ψ-RNA transcription kit, the INCOGNITO™ T7 5mC- and Ψ-RNA    transcription kit, the INCOGNITO™ T7 Ψ-RNA transcription kit, the    MESSAGEMAX™ T7 ARCA-capped message transcription kit, the SCRIPTCAP™    m⁷G capping system, the SCRIPTCAP™ 2′-O-methyltransferase kit,    SCRIPTGUARD™ RNase inhibitor, the SP6-SCRIBE™ standard RNA IVT kit,    the 17 mSCRIPT™ standard mRNA production system, and the T7-SCRIBE™    Standard RNA IVT Kit (all available on the web at www.cellscript.com    or from CELLSCRIPT, Inc., Madison, Wis., USA) and Monoclonal J2    Antibody and Monoclonal Antibody K1 (available from English    Scientific & Consulting, Szirák, Hungary) are incorporated herein by    reference.

1.-26. (canceled)
 27. A method comprising: a) performing in vitrotranscription (IVT) such that an IVT RNA composition is generated thatcomprises single-strand RNA (ssRNA) and double-stranded RNA (dsRNA)contaminants; b) contacting said IVT RNA composition with a prokaryoticendoRNase III to generate a reaction mixture, wherein magnesium cationsare present in said reaction mixture at a concentration of about 1-4 mM,and wherein said prokaryotic endoRNase III digests all, or substantiallyall, of said dsRNA contaminants to generate dsRNA digestion products;and c) purifying said ssRNA to generate a purified compositioncomprising said ssRNA, and wherein 0.05% or less of the mass of RNA insaid purified composition is dsRNA of 30 basepairs or longer.
 28. Themethod of claim 27, wherein said magnesium cations are present in saidreaction mixture at a concentration of about 1-2 mM.
 29. The method ofclaim 27, wherein said ssRNA comprises mRNA.
 30. The method of claim 29,wherein said mRNA encodes a human cell reprogramming factor protein or ahuman therapeutic protein.
 31. The method of claim 27, wherein saidssRNA comprises one or more modified bases.
 32. The method of claim 31,wherein said one or more modified bases are selected from the groupconsisting of: pseudouridine (ψ), 1-methyl-pseudouridine (m¹Ψ),5-methylcytidine (m⁵C), 5-methyluridine (m⁵U), 2′-O-methyluridine (Um orm^(2′-O)U), 2-thiouridine (s²U), and N⁶-methyladenosine.
 33. The methodof claim 27, further comprising: d) repeatedly transfecting human oranimal cells with said purified composition to cause a biological effectin said cells, without inducing significant cytotoxicity or cell deathin said cells.
 34. The method of claim 33, wherein said biologicaleffect comprises reprogramming said cells.
 35. The method of claim 34,wherein said ssRNA comprises mRNA encoding reprogramming factors. 36.The method of claim 34, wherein said ssRNA comprises mRNA that encodesat least one protein selected from the group consisting of: OCT4, SOX2;KLF4; LIN28; NANOG; MYC; c-MYC; c-MYC(T58A); and L-MYC.
 37. The methodof claim 27, wherein 0.01% or less of the mass of RNA in said purifiedcomposition is dsRNA of 30 basepairs or longer.
 38. The method of claim27, wherein 0.001% or less of the mass of RNA in said purifiedcomposition is dsRNA of 30 basepairs or longer.
 39. The method of claim27, wherein 0.0002% or less of the mass of RNA in said purifiedcomposition is dsRNA of 30 basepairs or longer.
 40. The method of claim27, wherein said ssRNA comprises a cap structure.
 41. The method ofclaim 27, wherein said ssRNA comprises a Cap I structure where the 5′penultimate nucleotide comprises a 2′-O-methyl-ribosyl group.
 42. Themethod of claim 27, wherein said ssRNA comprises a poly A tail.
 43. Themethod of claim 27, wherein said prokaryotic endoRNase III is from E.coli.
 44. The method of claim 27, wherein said dsRNA contaminants are ofa size greater than about 40 basepairs.
 45. The method of claim 27,wherein all, or substantially all, of said dsRNA contaminants aredigested by said prokaryotic endoRNase III to a size of about 12 to 15bp in length.